Induction of DNA damage, apoptosis and cell cycle perturbation mediate cytotoxic activity of new 5-aminosalicylate-4-thiazolinone hybrid derivatives.

Modulation of several targets in cancer cells enhances the effect of anti-cancer drugs. This can be achieved by using combinations of anti-cancer drugs or by designing new drugs with novel pharmacophore structures that target different molecules within cancer cells. We developed a panel of such compounds by accommodating two chemical entities (5-Aminoslicylic acid and thiazolin-4-one) known to have anti-cancer activities into a single framework structure. Using a panel of 7 cancer cell lines, two compounds (HH3 and HH13) showed efficient cytotoxic effects on some types of cancer comparable to the standard anti-cancer drug doxorubicin with tumor specificity and minimal effects on normal fibroblasts. Investigating the molecular mechanisms of the two compounds revealed (i) induction of DNA damage, (ii) cell cycle arrest in G2/M phase and (iii) induction of apoptosis as indicated by annexin-V staining and activation of caspases. These effects were more prominent in HH compounds-sensitive cells (with IC50 < 0.5µM) than -resistant or normal cells (with IC50 > 1µM). Moreover, both compounds modulate the expression and activity of several factors in the DNA damage response pathway (γ-H2AX, ATM, ATR, CHK1, CHK2), cyclins/cyclin dependent kinases and CDC25 phosphatase. Altogether, our results show that both HH3 and HH13 compounds are good candidates as anti-cancer drug leads for certain types of cancer and worth further detailed investigations of their safety and effectiveness on animal/xenograft models.

Interplay between Epigenetics, Expression of Estrogen Receptor- α, HER2/ERBB2 and Sensitivity of Triple Negative Breast Cancer Cells to Hormonal Therapy.

Triple negative breast cancer (TNBC) cells are resistant to hormonal/targeted therapies. This study aims to investigate epigenetic differences between TNBC and other types of breast cancer and the effect of epigenetic modulation on the response of TNBC cells to hormonal therapy. Thus, we investigated (i) the expression of different epigenetic markers, (ii) the effect of epigenetic modifying agents on the expression of ERα and HER2/ERBB2 and (iii) the effect on the response to tamoxifen in four breast cancer cell lines with different hormonal receptor status. Our results revealed a differential expression patterns of epigenetic markers in the four breast cancer cells. In TNBC cells, histone deacetylases (HDAC) 1 and 2 were less expressed, whereas HDACs 4 and 6 were overexpressed. Interestingly, treatment with epigenetic modifiers resulted in (i) a pronounced increase in the expression of ERα and HER2/ERBB2 along with (ii) an increase in the sensitivity of TNBC cells to tamoxifen. Collectively, this study indicates a different epigenetic background for TNBC cells, which represses the expression of ERα and HER2/ERBB2. Furthermore, we provide here the rationale for the use of epigenetic modifiers to enhance the response of TNBC to hormonal therapy through upregulation of ERα.

Inhibition of topoisomerase IIα sensitizes FaDu cells to ionizing radiation by diminishing DNA repair.

Despite the high efficiency of ionizing radiation (IR) to inactivate malignant tumours in general, an appreciable number of individual patients cannot be cured by standard IR. Head and neck tumours are not likely to be cured even by high-dose radiotherapy or chemotherapy. Accordingly, combined therapy is one of the most applicable strategies. Topoisomerase IIα is a ubiquitous enzyme that removes knots and tangles from the genetic material by generating and subsequently resealing of transient double-strand breaks. Due to its unique mechanism of action, topoisomerase IIα is the target of many chemotherapeutic agents such as etoposide. The aim of the present study is to examine the effect of inhibiting topoisomerase IIα by etoposide on the response of squamous cell carcinoma to IR. Results of the present study demonstrated a radiosensitizing effect for the topoisomerase IIα inhibitor etoposide on exponentially growing squamous cell carcinoma (FaDu) cell line especially at low radiation doses. This effect was found to be due to inhibition, by etoposide, of the repair of radiation-induced DNA damage. Cell cycle studies showed that the concentration of etoposide that sensitized the cells to radiation had no effect on the distribution of cells at different phases of the cell cycle. Synchronization of FaDu cells in different cell cycle phases revealed that proliferating G1 and G2 cells are responsible for sensitization of cells at low doses of ionizing radiation. It might, therefore, be concluded that topoisomerase II enzyme may be involved in the repair of radiation-induced DNA damage and consequently its inhibition constitute a strategy for sensitizing tumour cells to ionizing radiation.

Epigenetics and miRNA as predictive markers and targets for lung cancer chemotherapy.

Lung cancer cells show inherent and acquired resistance to chemotherapy. The lack of good predictive markers/novel targets and the incomplete understanding of the mechanisms of resistance limit the success of lung cancer response to chemotherapy. In the present study, we used an isogenic pair of lung adenocarcinoma cell lines; A549 (wild-type) and A549DOX11 (doxorubicin resistant) to study the role of epigenetics and miRNA in resistance/response of non-small cell lung cancer (NSCLC) cells to doxorubicin. Our results demonstrate differential expression of epigenetic markers whereby the level of HDACs 1, 2, 3 and4, DNA methyltransferase, acetylated H2B and acetylated H3 were lower in A549DOX11 compared to A549 cells. Fourteen miRNAs were dys-regulated in A549DOX11 cells compared to A549 cells, of these 14 miRNAs, 4 (has-mir-1973, 494, 4286 and 29b-3p) have shown 2.99 - 4.44 fold increase in their expression. This was associated with reduced apoptosis and higher resistance of A549DOX11cells to doxorubicin and etoposide. Sequential treatment with the epigenetic modifiers trichostatin A or 5-aza-2'-deoxycytidine followed by doxorubicin resulted in: (i) enhanced sensitivity of both cell lines to doxorubicin especially at low concentrations, (ii) enhanced doxorubicin-induced DNA damage in both cell lines, (iii) dysregulation of some miRNAs in A549 cells. In conclusion, A549DOX11 cells resistant to DNA damaging drugs have epigenetic profile and miRNA expression different from the sensitive cells. Moreover, epigenetic modifiers may reverse the resistance of certain NSCLC cells to DNA damaging agents by enhancing induction of DNA damage. This may open the door for using epigenetic profile/miRNA expression of some cancer cells as resistance markers/targets to improve response of resistant cells to doxorubicin and for the use of combination doxorubicin/epigenetic modifiers to reduce doxorubicin toxicity.

Induction and repair of DNA double-strand breaks using constant-field gel electrophoresis and apoptosis as predictive markers for sensitivity of cancer cells to cisplatin.

This study was designed to evaluate some parameters that may play a role in the prediction of cancer cells sensitivity to cisplatin (CIS). Sensitivity, induction and repair of DNA double-strand breaks (DSB), cell cycle regulation and induction of apoptosis were measured in four cancer cell lines with different sensitivities to CIS. Using a sulphorhodamine-B assay, the cervical carcinoma cells (HeLa) were found to be the most sensitive to CIS followed by breast carcinoma cells (MCF-7) and liver carcinoma cells (HepG2). Colon carcinoma HCT116 cells were the most resistant. As measured by constant-field gel electrophoresis (CFGE), DSB induction, but not residual DSB exhibited a significant correlation with the sensitivity of cells to CIS. Flow cytometric DNA ploidy analysis revealed that 67% of HeLa cells and 10% of MCF-7 cells shift to sub-G1 phase after incubation with CIS. Additionally, CIS induced the arrest of MCF-7 cells in S-phase and the arrest of HepG2 and HCT116 cells in both S phase and G2/M phase. Determination of the Fas-L level and Caspase-9 activity indicated that CIS-induced apoptosis results from the mitochondrial (intrinsic) pathway. These results, if confirmed using clinical samples, indicate that the induction of DNA DSB as measured by CFGE and the induction of apoptosis should be considered, along with other predictive markers, in future clinical trials to develop predictive assays for platinum -based therapy.

Antagonism between curcumin and the topoisomerase II inhibitor etoposide: a study of DNA damage, cell cycle regulation and death pathways.

UNLABELLED: The use of combinations of chemotherapy and natural products has recently emerged as a new method of cancer therapy, relying on the capacity of certain natural compounds to trigger cell death with low doses of chemotherapeutic agents and few side effects. The current study aims to evaluate the modulatory effects of curcumin (CUR), Nigella sativa (NS) and taurine on etoposide (ETP) cytotoxicity in a panel of cancer cell lines and to identify their underlying mechanisms. CUR alone showed potent antitumor activity, but surprisingly, its interaction with ETP was antagonistic in four out of five cancer cell lines. Neither taurine nor Nigella sativa affect the sensitivity of cancer cells to ETP. Examination of the DNA damage response machinery (DDR) showed that both ETP and CUR elicited DNA double-strand breaks (DSB) and evoked γ-H2AX foci formation at doses as low as 1 µg/ml. Cell cycle analysis revealed S phase arrest after ETP or CUR application, whereas co-treatment with ETP and CUR led to increased arrest of the cell cycle in S phase (MCF-7 cells) or the accumulation of cells in G 2/M phases (HCT116, and HeLa cells). Furthermore, cotreatment with ETP and CUR resulted in modulation of the level of DNA damage induction and repair compared with either agent alone. Electron microscopic examination demonstrated that different modalities of cell death occurred with each treatment. CUR alone induced autophagy, apoptosis and necrosis, whereas ETP alone or in combination with CUR led to apoptosis and necrosis. CONCLUSIONS: Cotreatment with ETP and CUR resulted in an antagonistic interaction. This antagonism is related, in part, to the enhanced arrest of tumor cells in both S and G 2/M phases, which prevents the cells from entering M-phase with damaged DNA and, consequently, prevents cell death from occurring. This arrest allows time for the cells to repair DNA damage so that cell cycle -arrested cells can eventually resume cell cycle progression and continue their physiological program.

Interaction of celecoxib with different anti-cancer drugs is antagonistic in breast but not in other cancer cells.

Celecoxib, an inhibitor of cyclooxygenase-2, is being investigated for enhancement of chemotherapy efficacy in cancer clinical trials. This study investigates the ability of cyclooxygenase-2 inhibitors to sensitize cells from different origins to several chemotherapeutic agents. The effect of the drug's mechanism of action and sequence of administration are also investigated. The sensitivity, cell cycle, apoptosis and DNA damage of five different cancer cell lines (HeLa, HCT116, HepG2, MCF7 and U251) to 5-FU, cisplatin, doxorubicin and etoposide±celecoxib following different incubation schedules were analyzed. We found antagonism between celecoxib and the four drugs in the breast cancer cells MCF7 following all incubation schedules and between celecoxib and doxorubicin in all cell lines except for two combinations in HCT116 cells. Celecoxib with the other three drugs in the remaining four cell lines resulted in variable interactions. Mechanistic investigations revealed that celecoxib exerts different molecular effects in different cells. In some lines, it abrogates the drug-induced G2/M arrest enhancing pre-mature entry into mitosis with damaged DNA thus increasing apoptosis and resulting in synergism. In other cells, it enhances drug-induced G2/M arrest allowing time to repair drug-induced DNA damage before entry into mitosis and decreasing cell death resulting in antagonism. In some synergistic combinations, celecoxib-induced abrogation of G2/M arrest was not associated with apoptosis but permanent arrest in G1 phase. These results, if confirmed in-vivo, indicate that celecoxib is not a suitable chemosensitizer for breast cancer or with doxorubicin for other cancers. Moreover, combination of celecoxib with other drugs should be tailored to the tumor type, drug and administration schedule.

Targeting DNA double-strand break repair: is it the right way for sensitizing cells to 5-fluorouracil?

Inhibition of the repair of 5-fluorouracil (FU)-induced DNA lesions may improve the response of many tumors to this anticancer agent. Despite the identified associations between DNA strand breaks and the lethality of thymidylate synthase inhibitors, the role of DNA double-strand break (DSB) repair pathways in a cellular response to 5-FU treatment has not been studied yet. Isogenic cell lines defective (irs1SF), wild type (AA8), or reconstituted (1SFK8) in the DSB repair protein XRCC3 were used to investigate the effect of defective DSB repair on the overall sensitivity of cells to 5-FU and to see how targeting DSB repair may affect other cellular responses to 5-FU. Treatment with 5-FU resulted in (i) similar induction of DSB in both cell lines as indicated by the formation of gamma-H2AX (a marker for DSB). The repair of these breaks was complete in AA8 but not in irs1SF cells. (ii) Concentration-dependent reduction in the survival of both cell lines. The AA8 cells were six times more sensitive to 5-FU than the irs1SF cells. (iii) An earlier and more prolonged G(1)/S phase arrest in AA8 compared with the irs1SF cells. (iv) Induction of apoptosis as indicated by sub-G(1) cells and caspase-3 activity in AA8 but not in irs1SF cells. XRCC3 complementation of irs1SF cells restored the wild-type phenotype. This result shows that targeting DSB repair is not always associated with increased sensitivity to DNA damaging agents such as 5-FU because it may affect other cellular responses such as cell cycle regulation and induction of apoptosis.

Altered expression of proliferation-inducing and proliferation-inhibiting genes might contribute to acquired doxorubicin resistance in breast cancer cells.

This study was designed to investigate the molecular changes that may develop during exposure of breast cancer cells to anticancer agents and that may lead to acquired resistance. We used two breast cancer cell lines, a parental (MCF7/WT) and a doxorubicin-resistant (MCF7/DOX) one. Cell survival, cell cycle distribution and RT-PCR expression level of genes involved in DNA damage response, MDR1, GST and TOPOIIalpha were measured. MCF7/DOX cells were five-fold more resistant to doxorubicin (DOX) than the MCF7/WT cells. DOX treatment causes arrest of MCF7/DOX cells in G1 and G2 phases of cell cycle whereas MCF7/WT cells were arrested in S-phase. The molecular changes in both cell lines due to DOX treatment could be classified into: (1) the basal level of p53, p21, BRCA1, GST and TOPOIIalpha mRNA was higher in MCF7/DOX than MCF7/WT. During DOX treatment, the expression level of these genes decreased in both cell lines but the rate of down-regulation was faster in MCF7/WT than MCF7/DOX cells. (2) The expression level of MDR1 was the same in both cell lines but 48 and 72 h of drug treatment, MDR1 disappeared in MCF7/WT but still expressed in MCF7/DOX. (3) There was no change in the expression level of BAX, FAS and BRCA2 in both cell lines. Conclusively, after validation in clinical samples, overexpression of genes like BRCA1, p53, p21, GST, MDR1 and TOPOIIalpha could be used as a prognostic biomarker for detection of acquired resistance in breast cancer and as therapeutic targets for the improvement of breast cancer treatment strategies.

Apoptosis is the most efficient death-pathway in tumor cells after topoisomerase II inhibition.

OBJECTIVE: To compare the efficiency of apoptosis and other modes of cell death in killing tumor cells after the induction of DNA damage by topoisomerase inhibitors like etoposide. METHODS: This study was carried out in the Tumor Biology Department, National Cancer Institute, Cairo University, Cairo, Egypt, from September 2005 to August 2007. The breast cancer MCF7, the cervix carcinoma, human cervical adenocarcinoma Hela, and the brain tumor U251 cell lines were exposed to etoposide. Apoptosis was detected using the flow cytometry and the DNA ladder formation methods. Cell viability was determined by a colorimetric assay, and the residual DNA double-strand breaks dsb were measured by gel electrophoresis. RESULTS: The Hela cells were the most, the MCF7's were moderately, whereas the U251's were the least sensitive to etoposide. Apoptosis was detected only in Hela cells whereas the other 2 cell lines showed a very low level of apoptosis only 3% increase above the control cells. At equitoxic drug concentrations namely IC50, the Hela cells showed the lowest amount of non-repaired DNA dsb, and the MCF7's showed the highest amount, whereas the U251 cells showed a moderate amount. CONCLUSION: These results indicate that although other modes of cell death exist, apoptosis is the most efficient and requires lower drug concentrations and fewer numbers of non-repaired dsb to give the same killing effect. Clinically, this means that tumors that can execute apoptosis may require lower doses of topoisomerase inhibitors than those that lost the ability to exercise apoptosis.

Misrejoined , residual double strand DNA breaks and radiosensitivity in human tumor cell lines.

PURPOSE: The aim of the present study is to investigate whether differences between tumor cells in radiosensitivity are related to misrejoined- or residual DNA-double strand breaks. MATERIAL AND METHODS: An assay that allows measurement of absolute induction frequencies for DNA double strand breaks (DSBs) in defined regions in the genome, and that quantitates rejoining of correct DNA ends has been used to study repair of DSBs in three human tumor cell lines. DNA double-strand breaks (DSBs) were measured within a 3.5-Mbp Not 1 fragment on chromosome X of human tumor cell lines with different radiosensitivities. Correct rejoining of DSBs was measured by hybridization of single-copy DNA probe to Not 1 restriction fragments separated according to size by pulsed field gel electrophoresis (PFGE). Induction of DSBs is quantified from the decrease in the intensity of the hybridizing restriction fragment and an accumulation of a smear below the band. Rejoining of DSBs results in reconstitution of the intact restriction fragment only if correct DNA ends are joined. By comparing results from this technique with results from a conventional electrophoresis technique (FDR assay) that detects all rejoining events, it was possible to quantitate the misrejoining frequency after 50Gy of X irradiation. Residual breaks were measured 24h after irradiation. RESULTS: In terms of clonogenic assay, squamous cell carcinoma cell line (4451) was the most radiosensitive, followed by the breast carcinoma cell line (BB) while the bladder carcinoma cell line (RT112) was the most radioresistant. Twenty-four hours after irradiation, 4451 cell line accumulated the highest level of residual (non-repairable) DSB followed by BB and then RT112 cell line, which showed the lowest level of residual DSB. This was the same rank as in the radiosensitivity assay. Regarding DSB misrejoining, RT112 cell line showed the highest percent of incorrectly repaired DSB, which does not agree with the results of the radiosensitivity assay. CONCLUSION: From our data, it could be concluded that residual (non repairable) DSB is more important in terms of radiosensitivity than incorrectly repaired DSB.

Loss of heterozygosity at BRCA1, TP53, nm-23 and other loci on chromosome 17q in human breast carcinoma.

BACKGROUND: In Egypt, breast cancer ranks number one among the female malignancies. Activation of oncogenes and inactivation of tumor suppressor genes are thought to play an important role in the development and progression of breast cancer. PURPOSE: The present study is a trial to investigate the role of chromosome 17 in sporadic invasive ductal carcinoma of the breast through detection of LOH for 6 highly polymorphic microsatellite loci, two of which are located at BRCA1 gene (D17S855 and D17S856), one at TP53 gene, one at nm-23 gene and finally two at 17q12-12.3 (D17S183 and D17S250). MATERIAL AND METHODS: Tissue samples and their corresponding safety margin normal tissues were collected from 25 patients with invasive ductal carcinoma of the breast of grades 2 and 3. LOH was detected for the 6 highly polymorphic microsatellite markers mentioned previously using PCR assay. RESULTS: The percentage of overall LOH recorded was 68% of the cases examined. The highest LOH recorded in D17S855 and D17S856 (43% and 32% respectively), both markers are located at BRCA1 gene, followed by 32% LOH in nm-23 gene. D17S183 and D17S250, which are localised telomeric and centromeric to BRCA1 gene, showed 24% and 28% LOH, respectively. The lowest percentage of LOH was observed in the TP53 gene (14%). No significant correlation was found between each of the six markers used and lymph node status, grade, or menopausal status. LOH at the nm-23 marker exhibited a significant association with lymph node involvement. CONCLUSION: It can be concluded from the present study that BRCA1 gene may be involved in carcinogenesis of some sporadic breast cancer cases. Deletion in nm-23 gene is associated with advanced stage of the disease. Finally, another gene located at 17q12-12.3 region may be involved in some sporadic breast cancer cases.