The present invention relates to a new medical use of metformin and epothilone A in treatment of neoplastic diseases. Both metformin and epothilone A are of a natural origin.

Metformin is a biguanide oral hypoglycemic agent and is widely used in the treatment of type 2 diabetes at every stage of the disease, as well as polycystic ovarian syndrome and metabolic syndrome (Huang et al., 2016). Diabetes is associated with increased risk of concomitant cancer of the liver, pancreas, kidneys, stomach, large intestine, endometrium, cervix, breast and ovary, as well as leukemia and non-Hodgkin's lymphoma. The hypoglycemic (antihyperglycemic) action of metformin is associated with a decrease in glucose absorption in the small intestine, increased glucose transport into cells, inhibition of gluconeogenesis and decrease of free fatty acids levels in plasma.

Recent studies have indicated that metformin inhibits cell proliferation in several human cancers, like pancreatic cancer, thyroid cancer, gastric carcinoma, endometrial carcinoma and HCC (Daugan et al., 2016; Wu et al., 2016). Various mechanisms for anticancer activity of metformin in different cancer types have been proposed, such as cytotoxic effects, apoptosis induction or immunomodulation (Chae et al., 2016; Park, 2015).

Epothilones were isolated from mycobacteria (slime bacteria - Myxobacteriae) Soranghim cellulosiim. Chemically they are macrolides composed of a 16-member ring and exhibit a taxane-like effect. Like paclitaxel (PTX) or docetaxel, hitherto used in ovarian cancer therapy, they stabilize microtubules and inhibit their depolymerization, and also generate reactive oxygen species (Zhao et al., 2015). The efficacy of epothilone A in ovarian cancer cells SK.OV-3 is well documented. In previous studies preceding the present invention it has been shown that the compound is nearly 6 times more cytotoxic than paclitaxel. The advantage of epothilone A is that, unlike PTX, it is not a substrate for glycoprotein P, so it is effective even in cells that are resistant to taxanes. This compound also exhibits strong proapoptotic properties (Rogalska et al., 2013). As a naturally occurring compound, there are fewer side effects than in case of other synthetic epothilone analogs (Cheng et al., 2008). The illustrating data are presented below in Table 1. Table 1.

Epothilones induce tubulin aggregation, cause mitotic arrest, and prevent microtubule depolymerization (Kumar et al., 2016). They are a newer class of microtubule- targeting agents than taxanes with higher potency (6-25-fold) in cancer cells, even with high levels of P-glycoprotein (multidrug resistance protein) (Bollag et al., 1995; Ganesan et al., 2016; Rogalska et al., 2013; Zhang et al., 2014). Epothilones competitively inhibit the binding of paclitaxel to microtubules (Sharma et al, 2013). Epothilones are 4-130-fold more potent than taxanes in HCC cell lines (Mok et al., 2006). Clinical studies of epothilones in other solid tumors, including lung, ovarian or breast cancers, have demonstrated the high potency of their anticancer activity (Campone et al., 2013; Chang and Wang, 2013; Nayak et al., 2015; Yardley et al., 2015).

Metformin obtained from Galega officinalis has also anti-atherogenic, antihypertensive and antitumor effects (Jakimiuk, 2008, Grzybkowska et al., 201 1). In 2005, Ewans and colleagues published a cohort study on patients with type 2 diabetes and treated with metformin. In that group reduction of the risk of cancer (odds ratio 0.79) when compared to the non-metformin population (Evans et al., 2005). Molecular mechanisms of metformin antitumor activity have not yet been fully understood, but in vitro and in vivo studies suggest that metformin is involved in the activation of the cellular signaling pathway LKB1/AMPK involving tyrosine kinase and protein kinase activated with adenosine monophosphate, and also in the inhibition of the threonine-serine mTOR protein kinase pathway (the so-called mammalian target for rapamycin), and that metformin consequently inhibits tumor growth and proliferation of cancer-altered cells (Dowling et al., 2007; Gotlieb et al., 2008; Zakikhani et al., 2006). Another suggested mechanism is the inhibition with metformin of cellular transformation with respect to tumor stem cells that are resistant to certain chemotherapeutic agents. Studies conducted on ovarian cancer cell lines have shown that metformin inhibits tumor cell proliferation by LKB1/AMPK pathway activation, mTOR inhibition, and reduced cyclicne Dl expression (Gotlieb et al, 2008; Rattan et al., 201 1 ; Shank et al., 2012). On the other hand, OVCAR-3 and OVCAR- 4 ovarian cancer studies have shown that metformin increases the expression of pro- apoptotic Bax and Bad proteins, which may contribute to the effective treatment of chemotherapy-resistant ovarian cancer (Yasmeen et al., 201 1 , Milewicz et al., 2013). The experimental data published so far indicate that with respect of the ovarian cancer cells metformin causes reduction of the proliferation rate, cell adhesion and rate of cancer malignancy. The experiments were carried out using S OV3, CAOV3, OVCAR3 and IGROV1 , VOSE, A2780, CP70, C200, OV202, PE01 and PE04 cell lines and observations were based on retrospective studies (Romero et al., 2012; Wu et al., 2012). It is postulated that the antiproliferative activity of metformin is mediated by the AMPK kinase-dependent and AMPK kinase-independent signaling pathways and that metformin is considered as a potential chemotherapeutic agent for the treatment of ovarian cancer (Romero et al., 2012). The use of metformin is also associated with side effects such as nausea, vomiting, diarrhea, abdominal pain, loss of appetite and lactic acidosis (DeFronzo et al., 2016; Thompson and Trujillo, 2016).

Resistance of human tumors to anticancer drugs is most often ascribed to gene mutations, gene amplification or epigenetic changes that influence the uptake, metabolism or export of drugs from single cells.

Ovarian cancer and liver cancer are solid tumors characterized by high mortality and poor response to therapy.

According to the statistics of World Health Organization hepatocellular carcinoma (HCC) is the second most common cause of cancer-related deaths worldwide (Gong and Qin, 2016). Treatment of HCC is more difficult than for other solid tumors, possibly because of its expression of multidrug resistance, the high diversity in the specific mechanisms involved in its carcinogenesis, and the presence of chronic liver diseases in the background. Many trials for the development of new treatments have been conducted since sorafenib, but the results have been disappointing, except for that of regorafenib (Moriguchi et al., 2016).

Ovarian cancer is also the most lethal gynecological malignancy and the second leading cause of cancer-related deaths among women worldwide. Due to the absence of specific symptoms at early stages and lack of means for early detection, the vast majority of ovarian cancer patients are often diagnosed at an advanced stage of the disease, with 60%-70% of patients having stage III— IV disease at diagnosis (Lanceley et al., 2017; Ren et al., 2016). The current standard of care for advanced ovarian cancer includes surgical debulking followed by platinum- and taxane-based chemotherapy.

Currently, the combination of drugs used in ovarian cancer is mainly carboplatin or cisplatin with cyclophosphamide. Cyclophosphamide was replaced over the years with paclitaxel or docetaxel. This technique is increasingly being supplemented with anthracyclines or bevacizumab. Bevacizumab is used in combination with carboplatin or paclitaxel. It has been reported that Phase II and Phase III clinical trials on the use of such compounds as sorafenib and sunitinibin inhibiting angiogenesis are at progress. These enzymes control the DNA repair process. Another potential candidate drug is poly(ADP- ribose)polymerase inhibitor - olaparib. Introduction of olaparip to the used drug combination increased the patient's lifespan by 3 months. Folic acid receptor antagonists (farletuzumab) are also used in the treatment of ovarian cancer. Ovarian cancer cells, as opposed to normal cells, are overexpressed by these receptors (Bukowska et al., 2016).

The first treatment cycle usually produces a positive response, however subsequent doses of chemotherapy do not produce the expected results due to the rapid development of multidrug resistance. The use of two drugs in treatment is effective provided that both drugs have different mechanisms of action and block the pathways responsible for the death of cancer cells. The benefit of combination therapy is also a lower number of side effects, although on the other hand, different compounds may result in interactions between them.

High mortality of liver and ovarian cancer increases the need to design more effective therapeutic strategies.

Recent gene expression profiling studies have suggested that microtubules are an important target for therapeutic intervention in HCC or ovarian cancer (Emmanuel et al., 201 1 ; Yu et al., 2016). Furthermore, several studies have demonstrated the involvement of the mammalian target of rapamycin (mTOR) pathway in resistance to microtubule- targeting chemotherapeutic agents (Broggini-Tenzer et al., 2015; Yoon et al., 2016). Metformin in turn, can lead to inhibition of mTOR (Hanna et al., 2012).

Thus far the use of a combination of metformin and epothilone A in treatment of neoplastic diseases has not been reported.

It is therefore the object of the present invention to provide a new hint to a treatment of various cancerous diseases, especially those frequently observed in diabetic patients, such as cancer of liver, pancreas, kidneys, stomach, large intestine, endometrium, cervix, breast and ovary, as well as leukemia and non-Hodgkin's lymphoma. It has been now unexpectedly confirmed by various tests and analysis of the detailed mode of combined action of antimicrotubule agents and metformin in hepatoma and ovarian cancer cells, that the combination is effective in therapy.

The main aims of the invention have been attained in accordance to the claims that follow after the present description. The invention is defined in the main independent claim and the preferred embodiments of the invention are defined in the dependent claims.

The present invention is particularly beneficial when a cocktail of the therapeutic compounds consisting of epothilone A and metformin is used in:

^■ a treatment of patients with type II diabetes and cancer of the liver, pancreas, kidney, stomach, large intestine, endometrium, cervix, breast and ovary, as well as leukemia and non-Hodgkin's lymphoma,

^■ a treatment of patients with solely cancer of the liver, pancreas, kidney, stomach, large intestine, endometrium, cervix, breast and ovary, as well as leukemia and non- Hodgkin's lymphoma (metformin has a stronger antitumor effect in S OV-3 ovarian cancer cells at normal sugar level - unpublished studies),

^■ a treatment of patients with multidrug resistance after classic ovarian cancer treatment.

The invention will be explained in details further down with reference to the attached drawings wherein:

Fig. 1 represents the analysis by flow cytometry of the cell-cycle distribution in HepG2 (A) and SKOV-3 cells (B) following incubation with the drugs in the presence or absence of the inhibitors; representative histograms of HepG2 (A-l) and SKOV-3 (B-l) DNA content after treatment with epothilone A, metformin or their combination for 48 h;

Fig. 2 A) presents HepG2 cells stained with Hoechst 33258 and propidium iodide visualized by fluorescence microscopy (Olympus 1X70, Japan; bar 50 μΜ); typical observations were given after 48 h of drugs; B) represents fractions of the cells in early and late apoptosis and necrosis at 48 h points following the treatment of HepG2 cells with epothilone A, metformin or their combination;

Fig. 3 A) presents SKOV-3 cells stained with Hoechst 33258 and propidium iodide visualized by fluorescence microscopy (Olympus 1X70, Japan; bar 50 μΜ); typical observations were given after 48 h of drugs. B) presents fractions of the cells in early and late apoptosis and necrosis at 48 h points following the treatment of SKOV-3 cells with epothilone A, metformin or their combination; Fig. 4 illustrates the effect of epothilone A and metformin on ROS production in HepG2 and S OV-3 cells (A-D); DCF fluorescence in the cells incubated 2 h with the tested compounds (C- control; A - epothilone A; M - metformin; A+M - epothilone A + metformin), resulting from ROS-mediated oxidation of the probe; blue color - control and drugs treated samples without inhibitors; the cells were analyzed under an inverted fluorescence microscope (Olympus 1X70, Japan; bar 50 μΜ);

Fig. 5 illustrates effects of epothilone A and/or metformin on Akt activation in HepG2 and SKOV-3 cells;

Fig. 6 illustrates increased genotoxic activity of epothilone A + metformin combination in HepG2 and SKOV-3 cells (A-D); DNA damage was measured by monitoring the percentage of DNA in the comet tail by use of the alkaline (pH >13) comet assay; at the same time, experiments were carried out on cells treated with epothilone A or metformin alone; the cells were preincubated with the test compounds in the presence of antioxidant NAC (3 mM) for 1 h at 37°C; blue color - control and drugs treated samples without inhibitors;

Fig. 7 illustrates endogenous oxidative DNA damage measured as the mean comet-tail DNA of HepG2 and SKOV-3 cells; Oxidative DNA damage was recognized by the DNA-repair enzymes endonuclease III (Nth) and formamidopyrimidine-DNA glycosylase (Fpg), and compared with appropriate controls, untreated with any enzymes; *P<0.05, significant differences between probes analyzed without and with DNA-repair enzymes;

Fig. 8 illustrates the effect of epothilone A and/or metforminon cell death; relative H2AX, PARP-1 and CASP3 mRNA expression in drug-treated and untreated hepatocellular and ovarian cancer cell lines after 48 h; Blue color - control and drugs treated samples without inhibitors;

Fig. 9 shows another possible mechanism of action of the present drug combination;

Fig. 10 presents benefits of use of the present drug combination;

Fig. 11 is a graphical abstract of the present invention.

Detail description of the invention

Studies and research performed by the co-Inventors leading to the present invention have shown the high antitumor efficacy of both epothilone A and metformin. A natural consequence of the findings was to conduct the study on combined effect of the two compounds in the treatment cancerous diseases. The subject matter of the studies performed and thus also the present invention is the new medical use of the two compounds: epothilone A and metformin administered together in treatment of cancerous diseases, in particular those suffered by type II diabetic patients, i.e. cancer of the liver, pancreas, kidney, stomach, large intestine, endometrium, cervix, breast and ovary, as well as leukemia and non-Hodgkin's lymphoma. The studies were directed also to a mechanism of the combined action of the two therapeutically active compounds. This goal was achieved by determining the cytotoxicity of cumulative effects of drugs, genotoxicity and proapoptotic properties.

Cell cultures: Effectiveness of the combination of the two therapeutically active compounds was tested on the human HepG2 HCC cells (ATCC HB-8065) and human S OV-3 ovarian adenocarcinoma cells (ATCC HTB-77) obtained between 2010 and 2013 from the American Type Culture Collection (ATCC; Rockville, MD, USA).

The newly received cells were expanded and aliquots of <10 passages were stored in liquid nitrogen. All cell lines were kept at low passage, returning to original frozen stocks every 6 months. During the study, cells were thawed and passaged within 2 months in each experiment. The cells were cultured in DMEM and RPMI 1640 with 10% heat- inactivated FBS, penicillin (10 U/ml) and streptomycin (50 μg /ml), and regularly checked for mycoplasma contamination. The cells were cultured under an atmosphere of 5% C0 ₂ and 95% air at 37°C.

Reagents: epothilone A, metformin, ribonuclease A and propidium iodide (PI) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). 2',7'-Dichlorodihydro- fluorescein diacetate (DCFH ₂ -DA), TRIzol Reagent, SA (NF-κΒ inhibitor) and TCN (Akt inhibitor that selectively inhibits cellular phosphorylation/activation of Akt 1-3) were also purchased from Sigma-Aldrich. AKT [pS473] kit was purchased from Invitrogen (Camarillo, CA, USA). High Capacity cDNA Reverse Transcription Kit was obtained from Life Technologies (Carlsbad, CA, USA). Real-Time 2xHS-PCR Master Mix SYBR A was from A&A Biotechnology (Gdynia, Poland). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 and fetal bovine serum (FBS) were obtained from Cambrex (Basel, Switzerland). Trypsin-EDTA, penicillin and streptomycin were acquired from Sigma- Aldrich.

Drug administration: Test compounds were dissolved in DMSO for epothilone A and TCN, and water for metformin and SA. They were stored frozen at -20°C and divided into small portions. Concentrated drug solutions were thawed immediately before use, diluted in PBS, and added to the cell culture medium at the final concentration. The IC50 for epothilone A and metformin in SKOV-3 cells was 20.4 nM (Rogalska et al., 2013) and 14 mM (Rogalska et al., 2014b), respectively, compared with 24.6 nM and 21 mM in HepG2 cells (Rogalska et al., 2016b). The IC50 value is a measure of toxicity of the drugs and is defined as concentration of the substance which reduces cell viability to half of the untreated control cells. The concentrations of drugs and inhibitors used for study were based on IC50 values. 5 mM SA and 10 μΜ TCN were chosen for all experiments, which corresponded to survival of ~80% relative to control untreated cells ( anai et al., 2012; Sliwinska et al., 2015). All experiments were performed with the following subtoxic concentrations of drugs selected in previous studies: epothilone A (10 nM), metformin (10 mM), epothilone A : metformin (10 nM : 10 mM).

In part of the experiments, some of the cells were preincubated with an antioxidant (3 mM NAC) for 1 h, and then the drugs were added at the appropriate concentrations and incubation was continued for the required period of time under the same conditions.

Cytotoxicity: Cytotoxicity of both the individual compounds and their combination was assessed by the MTT assay using a spectrophotometric method. The method involves measuring the reduction of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) (Sigma-Aldrich, Corp., St. Louis, MO, USA) to a colored (blue) formazan. When the tested compounds were added together - within the selected concentration range (below the IC ₅ 0 as defined above), the observed decrease in cell viability was greater than that noted for the individual compounds. It confirms that dosages of tested compounds may be reduced and thus the side effects of the drug combinations may also be reduced.

Mechanism of a combined action of drugs: To investigate the combined action of the drug combination, their influence on Akt kinase level was assayed using ELISA immunofluorescence method using specific monoclonal antibodies. Akt kinase is one of the proteins involved in apoptosis, which is probably affected by epothilones and metformin. It belongs to the proteins associated with the so-called pro-life pathways. A signal cascade, capable of enhancing cell survival, can potentially enhance its resistance to drug-induced apoptosis. Akt kinase also inhibits the cytochrome c outflow from mitochondria in a way independent of Bad phosphorylation. It is believed that the activity of the Akt kinase alone is not sufficient to initiate oncogenesis, while the enzyme may contribute to a tumor progression by inhibiting apoptosis and promoting appropriate changes in metabolism. The most recent studies show that, depending on the type of cells, individual Akt isoforms may have a positive or negative effect on the migration and invasion of tumor cells. Akt kinase is also involved in the regulation of the rumor angiogenesis process, and the increase of the enzyme phosphorylation level is associated with cell cycle disorders. The effect of Akt kinase (PKB) activity is an inhibition of apoptosis, stimulation of tumor cell proliferation, for example by induction of transcription factor NF-KB, which may lead to the expression of anti-apoptotic genes. Akt kinase levels were determined according to the Invitrogen protocol (Camarillo, USA) after 48 h incubation with drugs. It was observed that epothilone A and metformin led to a statistically significant increase in the Akt protein level. On the other hand, after the use of Triciribine inhibitor (TCN), in the epothilone A and metformin treated cells the activity of Akt decreased. When both tested compounds were administered at the same time, the activity of Akt increased in comparison to the control, and what was essential the combination of the drugs activated the Akt protein less than epothilone A alone, which is beneficial from the point of view of therapy. The TCN inhibitor led to a significant decrease in activity of the protein.

Cell cycle analysis: The cell cycle was analyzed using cytometric method. In the pool of properly proliferating cells (untreated with the tested chemical compounds), three main cell populations are distinguished G0/G1 , S and G2/M. In case of cells treated with cytotoxic drugs, an additional sub-Gl population is observed. Literature indicates that both tested drugs, due to their effect on the cell cycle regulating proteins, may interfere with the cell cycle normal course. It is suggested that epothilones among others lead to an increase of the p27 protein activity, which protein is an inhibitor of kinases. They also reduce the activity of the cyclin-dependent CDK2 kinase, weakening thereby phosphorylation of the Rb protein being necessary for the transition of cell transition from Gl phase to S phase. Cyclin A is related to both CDK2 and CDK1 kinases. It is suggested that epothilones and metformin may indirectly regulate Akt kinase activity. The now performed determinations have confirmed that the tested combination of drugs significantly increases the percentage of apoptotic cells in the sub-Gl phase in comparison to the individually used compounds. Additional sub-Gl growth was also observed when a combination of the drugs was pre- incubated with the NF- Β inhibitor or the Akt protein. The changes were dependent on duration of preincubation (studies were performed for 4, 24 and 48 hours). No epothilone- specific block in G2/M phase was observed. It may be due to the use of the subtoxic concentration of the compound. Metformin generated a distinct block in Gl phase.

ROS production: The level of reactive oxygen species was also monitored in SKOV-3 cell line and human HepG2 HCC cell line treated with the tested compounds. The changes were dependent on duration of incubation (2, 4, 24 h). The 2',7'-dichlorodihydro- fluorescein diacetate (DCFH2-DA) probe was used and its final concentration was 5 mM. DCFH2-DA after deacetylation to 2',7'-dichlorodihydro-fluorescein (DCFH2) is intracellularly oxidized to 2',7'-dichlorofluorescein (DCF). Kinetic changes of DCF fluorescence were monitored at the excitation wavelength λεχ = 485 nm and the emission wavelength em = 530 nm, using a fluorimetric plate reader (Fluoroskan Ascent FL., Labsystem Inc.). The individual compounds and their combination after 2 hours of incubation generated low ROS levels. However, a 30% increase in ROS levels were noted when combination of both tested compounds was preincubated with inhibitors of Akt and NF-KB proteins.

Comet assay: DNA damages were also assessed using a comet method. Two exemplary variants of the method were performed: alkaline version and oxidation version with enzymes: endonuclease III (ENDO III) recognizing apurinic/apirymidin sites and Fapy glycosylase (FPG) identifying formamidopyrimidines and 8-oxoguanine. The comet method allows for detection of single- and double-stranded DNA cracks, oxidative damages, pyrimidine dimers and photoproducts 6-4, as well as DNA-DNA and DNA- protein cross-linkages. Additionally, in the alkaline comet version, incubation with N- acetylcysteine antioxidant was used to estimate the contribution of reactive oxygen species to the generation of DNA damages. It was observed that the percentage of DNA in the tail was dependent on the time of incubation with the tested compounds. The highest changes were observed after 24 h incubation with combinations of the compounds with inhibitors of anti-apoptotic proteins (triciribine, sodium salicylate), using the alkaline variant. Moreover, the highest level of oxidative damages occurred also after the combination of tested compounds was used. The obtained results indicate the importance of DNA damages in the mechanism of action of the combination of the tested compounds. For this process the reactive oxygen species are co-responsible.

PCR analysis: PCR analysis was also performed. The level of mRNA for the H2AX gene involved in repair of the double-strand DNA cracks (double-strand breaks - DSB) was evaluated, as well as for the PARP-1 gene involved in the processes of DNA repair, detection and signaling of single and double DNA cracks. The level of mRNA for caspase-3, the executive enzyme of the apoptosis process, was also determined. The appearance of DSB in the cell nucleus triggers H2AX histone phosphorylation at Serl 39, and the phosphorylated H2AX is referred to as γΗ2ΑΧ. H2AX phosphorylation results in accumulation of repair agents at the site of DNA damage. It is considered to be a marker of apoptosis (Siddiqui et al., 2015). In turn, PARP-1 not only conducts the poly(ADP- ribosyl)ation reaction, but also directly interacts with DNA repair proteins. In the case of large number of DNA damages, strong activation of PARP-1 leads to depletion of the NAD+ and ATP pools, which depletion may lead in consequence to cellular malfunction and cell death by necrosis. Excessive activation of the PARP-1 enzyme caused by oxidative stress and genotoxic stress contributes to the pathomechanism of vascular diseases, neurodegenerative diseases, septic shock, type I diabetes, immune disorders, and also cancers (Olah et al., 2015). In order to prevent this process, epothilones lead to activation of caspase-3, which cleaves PARP-1, which is considered to be a marker of the apoptosis process (Rogalska and Marczak, 2015). It has been observed that both tested compounds increase PARP-1 and H2AX levels. The compounds used in combination, however, lead to a larger, statistically significant increase in the level of mRNA of the above indicated genes, which is indicative of increased cytotoxic and apoptotic potency of the drug combination.

Discussion: Assuming that the measure of the effectiveness of antitumor drugs is their cytotoxicity and the ability to induce apoptosis in cancer cells, it can be concluded that the tested compounds used in combination provide a very good solution in chemotherapy.

This explains why the combination of epothilone A + metformin is a better solution than the same but individually used compounds. The use of the Akt protein inhibitor further strengthens the effect of the drugs. It can be an alternative to currently used combination regimens in patients with chemotherapy resistance in ovarian cancer.

Two inhibitors were used in order to establish the mechanism of action of epothilone A and metformin combination. The first was sodium salicylate (SA), an inhibitor of nuclear factor (NF)-KB. SA is an example nonsteroidal anti-inflammatory drugs (NSAIDs). HCC frequently develops in the setting of chronic hepatitis and/or cirrhosis following viral chronic infection, and ovarian cancer risk increases with pelvic inflammatory disease (Rasmussen et al., 2016; Takeda et al, 2016). SA is widely used clinically for treatment of inflammation, and is established in anticancer therapy (Bandiera et al., 2016). Another inhibitor used was triciribine (TCN), which inhibits phosphorylation/activation of Akt 1-3. TCN significantly inhibits Akt phosphorylation at threonine-309 and serine-474, which are required for full activation of Akt. TCN is additionally used to enhance anticancer therapy (Kim et al., 2015).

In the current study, the antitumor efficacy of metformin in HCC and ovarian cancer was investigated; either alone or in combination with the next generation of microrubule-destabilizing agent, epothilone A. We focused on the cell cycle, DNA damage, apoptosis and phosphatidylinositide 3-kinase (PI3K)/Akt, which might be modulated by oxidative stress (Chetram et al., 2013). Akt is the key signaling molecule involved in regulating cell survival or apoptosis, proliferation and metabolism (Benbrook and Masamha, 201 1 ). Phosphatidylinositol-3,4,5-triphosphate formed by PI3K facilitates phosphorylation and activation of Akt (Rai et al., 2015). Akt is the most commonly activated signaling element in several human cancers (Ignacio et al., 2016; Wang et al., 2016). There is evidence that low levels of reactive oxygen species (ROS) can induce cell proliferation and genetic instability, which may contribute to the oncogenic phenotype of cancer cells. At high concentrations, ROS can promote cellular senescence and apoptosis, and therefore may function as antitumorigenic agents. Indeed, cancer cells frequently exhibit abnormal redox status associated with increased basal production of ROS, and thus cannot tolerate higher levels of free radicals. Therefore, the use of compounds that interfere in redox regulation is a promising strategy to target tumors selectively (Massaoka et al., 2012).

Taking into account that epothilones generate free radicals (Rogalska et al., 2014a; Rogalska et al., 2013), ROS production mediated by epothilone A or metformin was measured, and the effect of each drug alone with their combination in both cell lines was compared. The DNA-damaging potential of the metformin and epothilone A combination was investigated. The alkaline version (pH >13) of the comet assay in order to reveal DNA single-stranded breaks (SSBs) and double-strand breaks (DSBs) as well as alkali-labile sites was performed. The induction of oxidative damage in DNA by the drugs was checked in the presence of the antioxidant N-acetyl-cysteine (NAC). Oxidative DNA damage was estimated by using two specific enzymes, endonuclease III and formamidopyrimidine- DNA glycosylase (Fpg).

The genotoxic mechanism of combined action of metformin and epothilone A has not been investigated. DNA lesions related to cell death induced by both drugs were analyzed. The role of apoptotic cell death in response to chemotherapy is increasingly important. Epothilones or metformin induce several markers of apoptosis but their crucial common mechanism of action is unknown. The induction of cell cycle arrest was determined by means of flow cytometry. Also the mRNA expression of the apoptosis- related genes CASP3, PARP-1 and H2AX was analyzed. Proteolytically active caspase-3 can cleave several cellular proteins, such as poly (ADP-ribose) polymerase (PARP) and DFF (DNA fragmentation factor), resulting in the morphological features and DNA fragmentation of apoptosis (Cheng et al, 201 1). In contrast, poly-ADP-ribosylation of different nuclear acceptors by PARP-1 might be due to DNA SSBs and DSBs(Gerwing et al., 2016; Valenzuela et al., 2002).The formation of nuclear DSBs triggers phosphorylation of H2AX at Serl 39, which is named γΗ2ΑΧ. Increased expression of H2AX leads to p53 response, which blocks cell division and proliferation (Guimaraes et al., 2016).

The present invention is based on the demonstrated ability of metformin and epothilone A to exert a combined anticancer effect on HepG2 and S OV-3 cell lines. Previously, it was confirmed that concomitant metformin and epothilone A decreased cell viability more than when administered separately (Rogalska et al., 2016a).

Akt plays a critical role in controlling cell survival and apoptosis. It inhibits apoptosis directly through phosphorylation and inactivation of several proapoptotic targets, including Bad, forkhead transcription factors, c-Raf and caspase-9, or indirectly via activation of NF-κΒ and subsequent transcription of prosurvival genes.

The changes have been observed in the level of Akt after epothilone A treatment in both tested cell lines. It is believed that in rat aortic vascular smooth muscle cells, epothilone B does not increase the level of Akt (Lim et al., 2007). However, Akt is also involved in the cellular response to taxanes (Bergstralh and Ting, 2006). Akt phosphorylation in human ovarian carcinoma cell line IGROV-1 and human lung adenocarcinoma cell line H460 at Ser 473 was modulated by paclitaxel (Zuco and Zunino, 2008).

There are conflicting reports on the effects of metformin on the level of Akt. In accordance to the present invention, metformin caused an increase in the level of Akt, which was inhibited by TCN. Metformin induces Akt activation in cisplatin-treated U251 human glioma, C6 rat glioma, SHSY5Y human neuroblastoma and L929 mouse fibrosarcoma and HL-60 human leukemia cell lines (Janjetovic et al., 201 1). Metformin also regulates the level of Akt in breast cancer (Buac et al., 2013). Contrasting results suggest that metformin inhibits Akt phosphorylation in human endometrial stromal cells after stimulation with androgen and insulin (Ferreira et al., 2014).

Combined epothilone A + metformin activate Akt to a lesser extent than epothilone A alone. Decreasing levels of Akt may influence the increasing toxicity of epothilone A + metformin combination. It has been suggested that Akt isoforms show opposite functions in tumor initiation and growth(Lu et al., 2015).

ROS are produced in all mammalian cells as a result of normal cellular metabolism and partly due to activation of oxidant-producing enzymes in response to exogenous stimuli. One of the first manifestations of the apoptotic process, irrespective of the cell type, is disruption of mitochondrial membrane function (Redza-Dutordoir and Averill- Bates, 2016). Previously, it has been confirmed by the present Inventors that epothilone A or epothilone B generated ROS in S OV-3 cells (Marczak et al., 2014; Rogalska et al., 2013). The Inventors also detected an increase in ROS level after epothilone A treatment in HepG2 cells. Other data have confirmed that paclitaxel and docetaxel increased the formation of ROS in HepG2, Hep3B, HA22T/VGH and Hepa 1-6 hepatoma cell lines, however, antioxidant treatment did not block drug-induced cell cycle and growth inhibition effects (Lin et al., 2000). Some studies have demonstrated that silencing 5' AMP-activated protein kinase (AMPK) promotes the Warburg effect. As a result of AMPK activation, metformin can oppose the Warburg effect in favor of oxidative phosphorylation, and thus acts as a metabolic tumor suppressor(Daugan et al., 2016). Metformin is a partial inhibitor of complex 1 of the mitochondrial electron transport chain, which causes an abnormal flow of electrons to oxygen and leads to accumulation of ROS within the mitochondrial matrix. Metformin increased the level of ROS in MDA-MB-231 and MDA-MB-435 cells (Gao et al., 2016), OVCAR3, CAOV3 and SKOV3 ovarian cancer cells, and in PA-1 or liver cancer HepG2 and stem cells(Petrushev et al., 2012). This mechanism may provide an AMP -independent, upstream pathway for metformin to damage cancer cells through ROS production and mitochondrial damage (Chan and Miskimins, 2012; Ohnishi et al., 2016) .

The Inventors observed that combination of epothilone A + metformin significantly increased level of ROS. SA and TCN did not affect the level of ROS generated by epothilone A. In contrast, they raised the level of ROS mediated by metformin and epothilone A + metformin combination in both cell lines. The present results confirm that SA mediates ROS production. Generation of ROS is critical in triggering apoptotic tumor cell death through a Racl-NADPH oxidase-dependent pathway in gastric tumor cell line SNU-16 and human colon adenocarcinoma HT-29 and HCT1 16 cells. ROS are mediators of mitochondrial membrane potential collapse, which leads to the release of cytochrome c followed by caspase-9 and -3 activation, leading to apoptosis (Chung et al., 2003). Inhibition of NF- Β by SA reduces levels of ferrit in heavy chain in cutaneous T-cell lymphoma cells, thus increasing ROS levels and inducing apoptosis ( iessling et al., 2009).

The Inventors showed that epothilone A and metformin alone and in combination regulate apoptosis via ROS generation. The pathway of ROS-mediated Akt activation and subsequent apoptosis induction has been recently discovered (Jeong et al., 2015; Rai et al, 2015). Epothilone A and metformin activates Akt pathway and it might be important for regulating apoptosis of hepatoma or ovarian cancer cells. In addition, induction of ROS production by drugs might be upstream of the PI3K/Akt pathway because ROS production is apparently not inhibited by TCN.

In the present invention it is indicated that ROS are important mediators of cell death induced by epothilone A + metformin combination. This hypothesis is supported by experiments showing that NAC partially suppressed such changes.

The Inventors suggest that cell-cycle-regulating proteins are involved in the mechanism of action of epothilone A + metformin combination. It was also believed that evaluation of the sub-Gl population would reveal a correlation between apoptosis and cell cycle distribution(Son et al., 2016).

The present results were confirmed by double staining the compounds combination- treated cells with Hoechst 33258 and PI. The observed increase in the apoptotic cell fraction was mainly due to the direct interaction of the investigated drugs with nuclear DNA. Epothilone A + metformin combination caused the greatest increase in the apoptotic cell fraction, and the highest appearance of DNA damage.

The studies leading to the present invention showed that epothilone A did not block SKOV-3 or HepG2 cells in G2/M phase. This may have been because in the present experiments low, nontoxic doses of the drugs were used. It is known that epothilone B causes time-dependent growth of SKOV-3 and Hey ovarian cancer cells arrested at G2/M phase (Bukowska et al., 2016; Pellicciotta et al, 2013). Moreover, in SKOV-3 cells, epothilone B acts via a cyclin-dependent kinase (CDK)l/cyclin B-mediated pathway and has the ability to inhibit CDK1 indirectly (Bukowska et al., 2016). Paclitaxel and docetaxel have dose-dependent effects on hepatoma cell lines. Cells treated with a high dose (0.1 mM) of docetaxel or paclitaxel are arrested in G2/M phase, followed by generation of polyploidy or apoptosis; however, low dose (0.01 mM) treatment induces apoptosis without G2/M arrest (Lin et al., 2000).

The Inventors also observed that metformin reduced the number of cancer cells in Gl phase in a time-dependent manner. There was a parallel reduction in the percentage of cells in S and G2/M phases. Other studies have indicated that metformin initiates cell cycle arrest by reduction of cyclin like Dl , leading to dose-dependent inhibition of proliferation without inducing apoptosis (Daugan et al., 2016). Metformin exerts activity against HCC through inhibition of the mTOR translational pathway in an AMPK-independent manner. This leads to Gl phase arrest and subsequent apoptosis through the mitochondria-dependent pathway (Xiong et al, 2012).

According to the present invention epothilone A + metformin combination induces the highest growth of sub-Gl cells. The effect of the drug combination was enhanced by preincubation with SA or TCN. SA markedly increases the number of sub-GO/Gl cells. A similar effect has been reported for TCN (Marra and Liao, 2001 ; Rai et al., 2015). This leads to a dose-dependent increase in the number of cells in G0/G1 phase and decrease in S- and G2/M-phase cells, indicating cell cycle arrest. TCN also decreases cyclin Dl , similar to metformin (Gloesenkamp et al., 2012). Apoptosis-like morphological events, such as apoptotic body formation, alterations in the structure of the cell nucleus, were observed 48 h after epothilone A + metformin administration. The morphological changes were correlated with the DNA damage induced by this compounds by that time point. These observations give a new insight into the mechanism of action of epothilone A + metformin combination.

Mitochondrial homeostasis is critical in regulating ROS-mediated apoptosis. In many cases, these mitochondrial events are a prerequisite for the activation of a family of caspases that are important mediators of apoptosis. Activation of caspase-3 is the key intracellular molecules involved in apoptosis execution. Epothilone A and metformin in both types of tested cancer cells were involved in elevation of caspase-3 transcription. Similar results were obtained previously in S OV-3 cells treated by epothilone B (Rogalska et al., 2014c). Metformin in turn, induces expression of caspase-3 and the number of apoptotic signs in SCC9 cells (oral squamous cell carcinoma) (Guimaraes et al., 2016).

The Inventors observed significant differences between epothilone A, metformin and their combination in their effect on mRNA level. Epothilone A + metformin increased the level of caspase-3 ~2.74-fold in HepG2 cells and ~3.26-fold in SKOV-3 cells in comparison to either drug alone. It has been confirmed that SO induces apoptosis by increasing the level of caspase-3. Preincubation of cancer cells with SA enhances the combined effect of epothilone A and metformin. The present results are in agreement with other studies. SA (10 mM) induces caspase-3 activation and degradation of its substrate, PARP, in a variety of cancer cells, such as HCT1 16 colon carcinoma cells (Lee et al., 2003). Similar results have been obtained with CTCL cells (Braun et al., 2012).

In the studies supporting the present invention PARP-1 was measured as a marker of DNA fragmentation. It has been confirmed that epothilone A increased PARP-1 mRNA level in both tested cancer cell lines. Similar results have been obtained in S GT4 cells, in which paclitaxel induced apoptosis through activation of caspase-3 followed by PARP degradation (Kim et al., 201 1). Previously, the Inventors have established that epothilone B induces the mitochondrial release of cytochrome c in SKOV-3 and OV-90 ovarian cancer cells, and induces PARP cleavage activity of caspase-3 (Rogalska et al., 2014c; Rogalska and Marczak, 2015). It is also known that paclitaxel leads to PARP-1 cleavage in HCC cells (Xu et al., 2010)

The Inventors detected an increase in PARP-1 mRNA level in metformin-treated HepG2 cells. Other studies have reported significant cleavage activation of caspase-3 and PARP-1 in HepG2 cells (Xiong et al., 2012). Metformin induced low expression of PARP - 1 in ovarian cancer cells in the present study. However, expression of the cleaved forms of caspase-3 and PARP-1 was elevated by metformin treatment in A2780CP, A2780S, CI 3* and OV2008 ovarian cancer cell lines. The effect of metformin on DNA damage and repair seems to vary depending on the model tested(Li et al., 2012). Epothilone A and metformin in combination markedly increased the level of PARP-1 mRNA in both cell lines according to their sensitivity to each individual drug.

The increased level of ROS in cells induces DNA damage in the form of SSBs and DSBs, which in turn stimulate cells to activate several DNA repair mechanisms. Unrepaired and misrepaired SBs are serious threats to genomic integrity, such as chromosomal aberrations that simultaneously affect many genes and cause cell cycle arrest, induction of apoptosis, or mitotic cell death(Liubaviciute et al., 2015). Histone H2AX is a marker of DNA damage. In the present study, epothilone A increased H2AX mRNA level in both tested cancer cell lines in a manner dependent on the drug sensitivity of the cells. The Inventors observed an increase in the H2AX mRNA level after metformin treatment in SKOV-3 cells. In HepG2 cells, metformin induced a nonsignificant increase of H2AX mRNA. DNA of SCC9 cells treated with metformin under hypoxic conditions was more degraded than in control cells (Guimaraes et al., 2016). However, metformin did not induce a significant increase of H2AX mRNA in MiaPaCa-2 treated cells (Fasih et al., 2014). Differences in metformin action may be due to experimental variability and conditions.

The results obtained in the comet assay show that the DNA lesions included DNA SBs, alkali-labile sites and oxidative base damage. The Inventors observed that DNA damage decreased in the presence of the antioxidant NAC, which suggests that epothilone A and metformin, particularly in combination, induce ROS production and oxidative damage in DNA. Other research has shown that determination of free 8-hydroxyguanidine demonstrates that metformin increases oxidative DNA damage at concentrations of >30 μΜ in MCF-7 cells and at 1000 and 5000 μΜ in MDA-MB-231 cells (Marinello et al., 2016). It has been previously confirmed by the Inventors that epothilones induced DNA damage in ovarian cancer cells (Rogalska and Marczak, 2015).

The Inventors showed that combination of epothilone A and metformin potentiates DNA damage. A previous study showed that mice treated with paclitaxel plus metformin had a 60% reduction in tumor weight compared to controls, and the reduction was greater than that resulting from either drug alone(Lengyel et al., 2015). Metformin potentiates the effects of paclitaxel in endometrial cancer cells (Hanna et al., 2012), and sensitizes cancer cells to paclitaxel, carboplatin or doxorubicin. In combination with doxorubicin, metformin targets cancer stem cells and potentially leads to a decrease in the dose of chemotherapeutic agents (Hirsch et al., 2009).The potentiating effect of metformin on the activity of the various anticancer drugs has been confirmed in experiments with cisplatin in epithelial ovarian cancer cells (Diaz et al., 2005; Gotlieb et al., 2008). In contrast, adding microtubule-targeting agents (MTAs) to radiation, doxorubicin or etoposide leads to more sustained γ-Η2ΑΧ levels. It is also known that DNA-damage-repair proteins are transported on microtubules, and addition of MTAs sequesters them in the cytoplasm, explaining why combinations of MTAs and DNA damaging agents are common anticancer regimens (Poruchynsky et al., 2015).ROS inhibit the mTOR pathway via a mechanism that involves AMPK activation(Chan and Miskimins, 2012; Chen et al., 2010; Lennicke et al., 2015). Akt is a positive regulator of mTOR. Thus, the reduced expression of Akt by the combined action of epothilone A and metformin in the present study could have resulted in decreased mTOR activation, contributing to the elevated genotoxic effect and apoptotic changes in both tested cell lines. Such a hypothesis is supported by previous studies, in which metformin potentiated the effects of paclitaxel in endometrial cancer cells by mTOR inhibition (Hanna et al., 2012).

The results presently achieved clearly demonstrate that Akt activation by epothilone A or metformin does not lead to cell survival but rather it is a prerequisite for apoptosis. To the best of Inventors' knowledge, the finding supporting the present invention now presented represent the first report of the combination of epothilones and metformin causing apoptosis mediated by ROS. The effects of epothilone A+ metformin combination may be enhanced by NSAIDs or TCN. The Inventors showed that combination of epothilone A + metformin at low doses has a stronger antitumor effect when compared to either drug alone in the tested HCC and ovarian cancer models.

Further possible mechanism of action of the combination of epothilone A + metformin is illustrated in the accompanying drawing Fig. 9, while the benefits of use of the present drug combination is presented in Fig. 10.

The present results allow to expand the existing knowledge about combined action of epothilone A and metformin in cancer cell lines, and suggest molecular targets that can support the development of an effective therapy for solid tumors. Further work is warranted to determine the direct molecular and biochemical processes and exploit them for a potential therapeutic effect.

The following examples 1- 8 cover and explain the procedures used.

Example 1. Cell cycle analysis

Cellular DNA content was quantified by flow cytometry. Cells were treated with drugs for 4, 24 and 48 h. For experiments with inhibitors, cells were subjected to 1 h preincubation with TCN or SA. Epothilone A, metformin, or epothilone A + metformin combination was added and incubation continued for the required period of time under the same conditions. After incubation was completed, cells were collected, washed twice with PBS and fixed in 70% ethanol. After ethanol fixation (at least 24 h at 4°C), cells were washed in PBS and centrifuged at 7000 g for 10 min at 4°C. Pelleted cells were stained by adding 300 μΐ PBS containing PI and RNase at final concentrations of 75 μΜ and 20 μg/ml, respectively. This was followed by 1 h incubation in total darkness at 37°C. Stained cells were analyzed using a flow cytometer (Becton Dickinson, San Jose, CA, USA). The cell populations in particular phases of the cell cycle were quantified from a standard count of 10,000 cells by means of Flow Jo cytology software (Ashland, OR, USA).

Example 2. Morphological assessment of apoptosis and necrosis: double staining with Hoechst 33258 and propidium iodide

To determine the ratio between live, apoptotic, and necrotic cell fractions, simultaneous cell staining with Hoechst 33258 and PI was conducted. These fluorescent dyes vary in their spectral characteristics and ability to penetrate cells. The analysis was done with the Olympus 1X70 fluorescence microscope. Cells were cultured with the drugs for 48 h. Then, cells were removed from culture dishes by trypsinization, centrifuged and suspended in PBS at a final concentration of l xlO ⁶ cells/ml. One microliter of Hoechst 33258 (0.13 mM) and 1 μΐ of PI (0.23 mM) were added to 100 μΐ of cell suspension. The cells were then incubated at room temperature for 10 min in total darkness. At least 300 cells were counted on each slide and each experiment was done in triplicate. The percentages of particular cell types were determined from the total number of cells.

Example 3. Measurement of ROS production

Production of ROS was measured by DCFH ₂ -DA assay (Halliwell and Whiteman, 2004; Hempel et al., 1999). Intracellular ROS level was determined directly in cell monolayers in 96-well microplates using a Fluoroskan Ascent FL microplate reader (Labsystems, Stockholm, Sweden). HepG2 and S OV-3 cells in complete medium were incubated with appropriate concentrations of epothilone A and metformin for 2-48 h in the presence or absence of antioxidant NAC. To determine the production of ROS, cells were treated with 5 μΜ DCFH ₂ -DA at 37°C for 30 min, and the fluorescence of DCF was measured at 530 nm after excitation at 485 nm (DCFH ₂ -DA, after deacetylation to DCFH2, is oxidized intracellularly to its fluorescent derivative, DCF). Assays were performed in modified Hank's buffered salt solution (140mM NaCl, 5mM KC1, 0.8mM MgCl ₂ , 1.8mM CaCl ₂ , ImM Na ₂ HP0 ₄ , lOmM HEPES and 1% glucose, pH 7.0, without phenol red).

Example 4. Measurement of Akt protein

Cells were plated (5x 10 ⁵ ) in 5 ml culture medium in 60-mm Petri dishes. After 24 h, drugs or TCN was added and the cells were incubated for 48 h. The cells were washed in PBS and re-suspended in ice-cold cytosol extraction buffer containing phenylmethane- sulfonyl fluoride and protease inhibitor cocktail. Cell lysate was centrifuged at 10,000 g for 30 min at 4°C. The supernatant (cytosolic fraction) was collected and stored at -80°C. The Petri dish wells were coated with monoclonal antibody specific for Akt (regardless of phosphorylation state). During the first incubation, the Akt antigen from tested probes was bound to the immobilized antibody. After washing, a rabbit antibody, specific for Akt phosphorylated at serine 473, was added to the wells. After removal of excess detection antibody, a horseradish-peroxidase-labeled anti-rabbit IgG (immunoglobulin G) was added. The intensity of this colored product was directly proportional to the concentration of Akt present in the probes. The reaction was stopped by addition of 2 M sulfuric acid, generating a yellow color that was recorded at 450 nm with a PowerWave microplate reader (BioTek, Winooski, VT, USA).

Example 5. Comet assay

The comet assay was performed under alkaline conditions according to the procedure of Singh et al. (1988) with minor modifications in order to examine the extent of DNA damage. This version of the technique recognizes SSBs and DSBs, as well as the alkali-labile sites. The control and treated cells were collected at 4, 24 and 48 h after culture initiation (Singh et al., 1988). In some samples, the cells were preincubated with NAC to analyze the contribution of free radicals to DNA damage induction. Cells treated with 10 μΜ hydrogen peroxide for 10 min at 4°C served as a positive control. The cells were suspended in 0.75% low-melting-point agarose in PBS, pH 7.4. Next, this suspension (50 μΐ) was spread on frosted microscope slides precoated with a layer of 1% normal melting agarose. After gelling, the slides were treated with a lysis buffer consisting of 2.5 M NaCl, 100 mM EDTA, 1% Triton X-100, 10% DMSO and 10 mM Tris, pH 10, at 4°C for 1 h. The slides were placed in an electrophoresis solution (300 mM NaOH, 1 mM EDTA, pH >13) for 40 min to allow for DNA unwinding. Electrophoresis was carried out at 29V, 30 mA for 20 min. The slides were stained with 2 μg/ml DAPI (4',6-diamidino-2- phenylindole). All these steps were performed in total darkness to prevent additional DNA damage. Fifty randomly selected cells from each slide were measured using image analysis (Nikon, Tokyo, Japan) attached to a COHU 4910 video camera (San Diego, CA, USA), which was equipped with a UV-1 filter block consisting of an excitation filter (359 nm) and barrier filter (461 nm) connected to the image analysis system (Lucia-Comet v. 4.51 ; Prague, Czech Republic). For a direct comparison of the influence of epothilone A or metformin on DNA damage, the percentage of DNA in comet tails was estimated. In the samples preincubated with NAC, the level of DNA damage associated with free-radical formation was determined.

Example 6. Endonuclease assay

Endogenous and exogenous oxidative DNA lesions after treatment of HepG2 and S OV-3 cells were investigated. According to the standard comet assay, slides after lysis were washed three times in an Fpg Nthl buffer (40 mM HEPES-KOH, 0.1 mM C1, 0.5 mM EDTA, 0.2 mg/ml BSA, pH 8.0, and agarose); covered with 25 ml buffer or Nthl as well as Fpg at 1 mg/ml in buffer; sealed with a cover glass; and incubated for 30 min at 37°C. Further steps were performed as described in Example 4. The results obtained for an enzyme (Nthl or Fpg)were corrected by subtracting the level of DNA damage observed for the negative control. To check the ability of the enzymes (Nthl/Fpg) to recognize oxidized or alkylated DNA bases under the experimental conditions, the cells were incubated with hydrogen peroxide, lysed and treated with Nthl or Fpg.

Example 7. RNA isolation and cDNA synthesis

Total RNA was isolated using TRIzol Reagent and quantified spectrophotometrically. Concentration of RNA was measured with a Nanodrop system (BioTek). For this, RNA was diluted in water, and the absorbance was read at 260 nm against water as a blank. First-strand cDNAs were obtained by reverse transcription of 2 μg total RNA using High Capacity cDNA Reverse Transcription Kit (Life Technologies). cDNAs were stored at -20°C.

Example 8. Quantitative real-time reverse transcription polymerase chain reaction (RT-PCR)

Real-time gene expression analysis of target genes PARP-1, CASP3 and H2AX was performed using Real-Time 2*HS-PCR Master Mix SYBR A (A&A Biotechnology). The HPRT1 gene was used as an internal control. Allele-specific oligonucleotides listed in Table 2 were used as primers.

Table 2. Sequences of primers used for RT-PCR

Each PCR was performed in a 10 μΐ volume that included 5 μΐ RT HS-PCR MasterMix, 2 μΐ water-diluted cDNA template (50 ng), 1 μΐ each primer, and 1 μΐ water. The following PCR program was used: 95°C for 3 min and 40 cycles under cycling conditions: 95°C for 30 s, 60°C 30 s and 30 s at 72°C. Quantitative RT-PCR was carried out using the Eco Real-Time PCR System, Illumina (San Diego, CA USA). Relative RNA quantification was performed using the ACt method. ACt (Ct _{/a ge/ gene} - QHPRTI) values were recalculated into relative copy number values (number of target gene mRNA copies per 1000 copies of HPRTlmKNA).

Statistical analysis: The data are presented as means ± S.D. ANOVA was performed with the Tukey post hoc test for multiple comparisons (StatSoft, Tulsa, OK, USA). The significance level was set at a P value of 0.05. Significant differences are indicated in the figures as follows: (*) P<0.05 between samples incubated with the drugs in comparison to control cells (Fig. 7 - significant differences between probes analyzed without and with DNA-repair enzymes); (/) P<0.05 between samples incubated with the drugs (control, C; epothilone A, A; metformin, M) and SA; (o) P<0.05 between samples incubated with the drugs and TCN; (+) P<0.05 between samples incubated with the drug combination and epothilone A or metformin; (#) P<0.05 between samples incubated with drug combination + inhibitor (SA or TCN) and drug combination; and (·) PO.05 between samples preincubated for 1 h with 3 raM NAC and then incubated with epothilone A or metformin. All values are given as the mean ± S.D. of three independent experiments.

Further examples 9-14 present and discuss results of the experiments performed with reference to the drawings graphically presenting the results.

Example 9. Cell cycle distribution

Figs. 1 present the effect of epothilone A, metformin and epothilone A + metformin on the cell cycle distribution of HepG2 and SKOV-3 cells. The cells were treated with drugs for 4, 24 or 48 h and analyzed by flow cytometry.

Fig. 1 A shows a marked decrease in the number of HepG2 cells in Gl phase at 4 h after treatment with epothilone A + metformin with or without SA. There was also a significant rise in the number of cells arrested in G2/M phase. Single drugs did not affect the cell cycle phases. SA or TCN with epothilone A decreased the number of cells in Gl phase and increased those in G2/M phase. A similar effect was observed with the addition of metformin. TCN led to a reduction in the number of cells in the Gl , S and G2/M phases and preincubation with SA alone decreased the number of cells in S phase.

After 4 h incubation of SKOV-3 cells with the tested compounds, there was no difference in comparison to the controls (Fig. 1 B).

After 24 h, only treatment with epothilone A + metformin increased the number of cells in G2/M phase from 19% to 26% in HepG2 cells (Fig. 1 A). Epothilone A did not block G2/M phase. After 24 h, the proportion of apoptotic cells (sub-Gl phase) was ~8% after epothilone A treatment and ~15% after epothilone A + metformin treatment. It is worth mentioning that both inhibitors significantly increased the number of apoptotic cells after treatment with the drug combination. TCN raised the number of apoptotic cells induced by epothilone A.

After 24 h, the proportion of apoptotic SKOV-3 cells (sub-Gl phase) was -7% after epothilone A treatment and -18% after epothilone A + metformin treatment. TCN raised the proportion of apoptotic cells induced by epothilone A + metformin to 26.73% (Fig. 1 B). Metformin increased the proportion of cells in Gl phase from 48 to 61%. Epothilone A did not block Gl or G2/M phase.SA increased the number of cells in Gl phase (induced by epothilone A + metformin) and S phase (induced by metformin). TCN increased the number of cells in Gl phase after treatment with epothilone A and epothilone A + metformin.

After 48 h incubation of HepG2 cells (Fig. 1 A, 1 A-l), epothilone A + metformin increased the proportion of cells in the sub-Gl population to -28%. TCN increased the effects of the drugs alone and in combination (47.25%). At this time, epothilone A + metformin also stopped the cell cycle at the G2/M phase (42.37%).

In the case of S OV-3 cells, we observed that all treatments increased the number of cells in sub-Gl phase. The highest level of 27% was detected after epothilone A + metformin treatment. SA and TCN decreased the number of epothilone A-treated apoptotic cells. SA increased the numbers of metformin- and SA-treated cells, and TCN increased the number of epothilone A + metformin-treated cells (Fig.l B, 1 B-l). Metformin alone led to mitotic bloc in Gl phase.

To summarize, epothilone A, metformin and epothilone A + metformin induced apoptosis, which was most significant after 48 h treatment with epothilone A + metformin. The fraction of Gl cells after treatment with either drug alone increased in a time- dependent manner, whereas combination treatment increased G2/M mitotic arrest after preincubation of cells with SA in HepG2 cells.

Assessment of apoptosis and necrosis: double staining with Hoechst 33258 and propidium iodide: The ability of epothilone A, metformin and compounds combination to induce apoptosis or necrosis was evaluated by treating cells for 48 h. Then, morphological changes in SKOV-3 and HepG2 cells were analyzed by doublestaining with Hoechst 33258 and propidium iodide. These fluorescent dyes emit various types of fluorescence and differ in their ability to penetrate cells (Rogalska et al., 201 1 ). Thus, its uptake indicates loss of membrane integrity, characteristic of late apoptotic and necrotic cells(membrane-altered cells). Numerous changes in cell morphology, typical either for apoptosis or necrosis were detected (Fig. 2 A and Fig. 3 A). Alterations in the structure, size and shape of the cell nucleus were observed. DNA staining with certain dyes allows the direct observation of increasing the density of chromatin and shrinking the nucleus. Apoptotic cell loses a large quantity of water. This leads to wrinkling and shrinking of the cell membrane which leads to the small bumps on the surface of the so-called "blebs". There was observed a fragmentation of the nucleus and the cytoplasm. The formation of apoptotic bodies and cells disintegration were also presented.

Quantitative analysis of the fractions of early apoptotic, late apoptotic, and necrotic cells, is exhibited in 2B and 3B. The statistically significant changes were noted after HepG2 cells treatment with epothilone A (10% of the apoptotic cells). Epothilone A + metformin increased the number of apoptotic cells to -28%. TCN increased the effects of the drugs in combination (-49 % of apoptotic cells) (Fig. 2 B).

Similar changes were noted also after S OV-3 cells treatment with epothilone A (18% of the apoptotic cells), Fig. 3 B. Epothilone A + metformin increased the number of apoptotic cells to -24%. TCN and SA increased the effects of the drugs in combination (-38% of apoptotic cells). TCN increased also number of necrotic cells in combination treated cells to 36%.

Example 10. Measurement of ROS production

ROS production was examined as an index of oxidative stress during incubation of HepG2 and SKOV-3 cells with epothilone A, metformin and epothilone A + metformin in the presence or absence of NAC, SA or TCN, using the fluorescence probe DCFH ₂ -DA. Fig. 4 presents data expressed as a percentage of DCF fluorescence intensity in cells incubated with the drugs for 2-24 h. The intensity of DCF fluorescence in control cells measured after 2, 4 and 24 hwas taken as 100%. Our results clearly demonstrated that epothilone A + metformin treatment of HepG2 cells generated higher levels of ROS than single drugs. A maximum level of ROS was observed after 2 h treatment with epothilone A + metformin(l 15%) (Fig. 4 D). When the cells were preincubated with NAC, the level of ROS was significantly decreased in comparison to the control probes for all the tested times of incubation. SA and TCN increased the level of ROS in metformin-treated cells after 4 h incubation. TCN increased the level of ROS additionally after 24 h incubation with epothilone A + metformin.

Epothilone A + metformin treatment of SKOV-3 cells also generated a higher level of ROS than single drugs did. The maximum level of ROS was observed after 2 h treatment with epothilone A + metformin(125%) (Fig. 4 D). TCN and SA increased the level of ROS after 24 h incubation with metformin.

Microscopic examination of cells stained with H ₂ DCF-DA revealed increased DCF fluorescence in drug treated cells resulting from ROS-mediated oxidation of the probe.

When the cells were preincubated with NAC, the level of ROS was significantly decreased in comparison to the control probes for all the tested times of incubation. After 4 and 24 h of incubation, ROS were not detected in epothilone A- or metformin-treated cell lines. Preincubation with inhibitors of antiapoptotic proteins enhanced the effect of the tested compounds. The early increase in ROS formation suggests that the generation of ROS is an important mechanism of action that might contribute to the cytotoxicity of epothilone A + metformin in liver and ovarian cancer cells.

Example 11. Modulation of Akt

The level of Akt, a well-known cell survival signal protein, in HepG2 and S OV-3 cells treated with epothilone A, metformin or TCN were examined. TCN is tricyclic purine nucleoside analog that is metabolically activated inside cells by adenosine kinase to its mono-phosphate active analog TCN-P. TCN-P interacts with the PH domain of Akt and interferes with its membrane localization, thereby preventing Akt phosphorylation and subsequent activation (Sampath et al., 2013). We evaluated changes in Akt level after treatment with epothilone A + metformin. In some experiments, cells were preincubated with TCN (Fig. 5).

Epothilone A enhanced Akt phosphorylation at Ser-473 in a significant manner in both cell lines (HepG2, 388.71% and SKOV-3, 202%). Metformin also increased the level of Akt in HepG2 and SKOV-3 cells (~1.5-fold). When both drugs were given together, their effects were different. Epothilone A combined with metformin increased the level of protein, but growth was lower than in cells treated only with epothilone A (~3-fold in HepG2 and ~1.8-fold in SKOV-3 cells). Akt level in cancer cells corresponded to cell drug sensitivity. Preincubation with TCN resulted in a substantial decrease in Akt phosphorylation. These data indicate that epothilone A + metformin increase the sensitivity of both types of cancer cells to chemotherapy.

Example 12. Drug-mediated DNA damage

The percentage of DNA fragments in the comet assay, which is one of the methods for confirming death by apoptosis(Hunakova et al., 2014; Stefanou et al., 2015) was measured. The mean DNA damage was dependent on the cell type and duration of treatment with epothilone A, metformin or epothilone A + metformin. There was an increase in the percentage of DNA in the comet tails for ovarian and liver cell line cells as early as 4 h after treatment. The comet assay clearly demonstrated that epothilone A, similarly to metformin, induced DNA damage in HepG2 and SKOV-3 cells (Fig. 6). The increase in DNA damage after 4-48 h of incubation with epothilone A + metformin was significant. The highest percentage of DNA in the comet tails was observed after 48 h of epothilone A incubation in HepG2 (24%) and SKOV-3 (28%) cells (Fig. 6 B). Metformin generated a similar level of DNA damage after 24 and 48 h (-20%) in HepG2 cells and -30% in SKOV-3 cells (Fig. 6 C). Significant changes in the extent of DNA damage induced by metformin and epothilone A alone and in combination were detected at 4 h after exposure. The greatest differences between the single drugs and their combination were observed after 24 and 48 h incubation. At these time points, ~2.5-fold and ~2.7-fold increases in the percentage of DNA in the comet tails were noted in the epothilone A + metformin-treated cells, compared with the respective values for cells incubated with either drug alone (Fig. 6 D).

SA decreased the level of DNA damage induced by epothilone A in S OV-3 cells at all incubation times. However, we observed an increase in genotoxicity after 24 h combination treatment, when SKOV-3 cells were pretreated with both SA and TCN. The results indicate the participation of the antiapoptotic pathway.

To confirm whether ROS are mediators of DNA damage induced by epothilone A, metformin or epothilone A + metformin, the cells were treated with 3 mM NAC. Pretreatment of cells with the antioxidant diminished the level of DNA fragmentation. Generation of ROS is an important mechanism that might contribute to the genotoxicity of individual drugs and especially drugs in combination.

Example 13. Oxidative DNA damage

Fig. 7 shows endogenous oxidative DNA damage measured as the mean level of comet-tail DNA in HepG2 and SKOV-3 cells. Cells were lysed and treated with the enzymes endonuclease III (Nth) or Fpg. Nth converts oxidized pyrimidines into strand breaks. Fpg is involved in the first step of the base excision repair to remove specific modified bases from DNA, creating apurinic or apyrimidinic sites. The enzyme excises mainly 2,6-diamino-4-hydroxy-5- V-methyl formamidopyrimidine and 8-hydroxyguanine (Mrowicka et al., 2015; Poplawski et al., 2006).

The extent of oxidative DNA damage recognized by Nth and Fpg in human hepatocellular cancer cells after treatment with epothilone A + metformin was -10% higher than in cells that had not been postincubated with both enzymes. Similar results were obtained in SKOV-3 cells, however, epothilone A induced DNA damage recognized by Fpg at the level of 53.8% after 48 h of incubation. A higher level of DNA damage recognized by enzymes was observed in cells treated with epothilone A + metformin than after incubation with either drug alone. In HepG2 cells, a high level of DNA breakage was observed after incubation for 48 h with epothilone A + metformin, as shown by the high levels of endogenous oxidative damage to purine (74.6%) and pyrimidine (80%) bases. The most significant difference in the level of DNA fragmentation caused by epothilone A + metformin and visible with Fpg was observed after 48 h treatment of SKOV-3 cells (87.9%). Pretreatment of cells with Akt inhibitor increased the level of DNA lesions in HepG2 cells recognized by Fpg even after 4 and 24 h of incubation with epothilone A + metformin. Similar results were obtained for SKOV-3 cells. Pretreatment of cells with Akt or NF-KB inhibitor followed by epothilone A + metformin treatment in the presence of the inhibitor increased the level of DNA lesions recognized by Fpg after 4 h incubation, and by both enzymes after 24 h incubation. Prolonged incubation of cells with the drugs combination (48 h) leads to the considerable difference between the level of oxidized purines in HepG2 cells

Example 14. Effect of epothilone A and metformin on expression of genes involved in apoptosis

Changes in the levels of mRNA expression of genes associated with apoptosis and DNA damage, PARP-1, CASP3 and H2AFX, were analyzed using RT-PCR (Fig. 8). Epothilone A and metformin increased caspase-3 mRNA level in both cell lines. The effect was greater whenthe drugs were administered in combination rather than individually. Epothilone A + metformin increased the level of caspase-3 ~3.49-fold in HepG2 cells and ~3.33-fold in SKOV-3 cells. Changes were in agreement with drug cytotoxicity. SA and TCN also increased mRNA level of serine protease. Additionally, SA and TCN increased the effect of drug combination in HepG2 cells.

The H2AX mRNA level was markedly higher in SKOV-3 cells than in the control cells. We observed a significant ~1.25-fold increase in H2AX mRNA level in cells incubated with epothilone A for 48 h and an ~1.7-fold increase after metformin treatment (PO.05; Fig. 8). Epothilone A resulted in a similar increase in HepG2 cells (1.2-fold), however metformin did not generate significant changes. Combination of the drugs in both cell lines increased expression of H2AX mRNA level ~1.4-fold in HepG2 cells and ~3- fold in SKOV-3 cells. There was a significant difference between using single and combined drugs. The inhibitors did not show any changes in cells treated with epothilone A + metformin.

There were significant increases of ~1.6-fold and ~1.19-fold of PARP-1 mRNA level in the HepG2 cells incubated with epothilone A and metformin, respectively (P<0.05). Similar results (~1.25-fold increase) were detected in SKOV-3 cells treated with epothilone A. Metformin did not affect the level of PARP-1. However, in ovarian cancer cells, we observed that epothilone A + metformin combination led to the highest level of PARP-1 mRNA. Lesser changes were induced by drug combination in HepG2 cells; ~1.83-fold in comparison to the control cells. Preincubation of SKOV-3 cells with SA or TCN had no effect on level of PARP- 1 mRNA in cells treated by epothilone A, but both inhibitors increased gene expression in metformin-treated cells. Only preincubation with TCN elevate level of PARP- 1 mRNA in epothilone A and metformin treated cells. In HepG2 cells preincubated with SA or TCN and then treated with epothilone A, there was a decrease in PARP- 1 mRNA level, in contrast to cells treated with metformin. In S OV-3 cells, TCN pretreatment followed by epothilone A + metformin treatment increased mRNA levels of PARP- 1 .

Abbreviations:

DCFH ₂ -DA - 2',7'-dichlorodihydrofluorescein diacetate;

HepG2 - hepatocellular liver carcinoma cell line;

MET - metformin;

NAC - jV-cetylcysteine;

NF-KB - nuclear factor kappa B;

ROS - reactive oxygen species;

SKOV-3 - ovarian adenocarcinoma;

SA - sodium salicylate;

TCN - triciribine.