Loss of miR-101-3p Promotes Transmigration of Metastatic Breast Cancer Cells through the Brain Endothelium by Inducing COX-2/MMP1 Signaling.

Brain metastases represent one of the incurable end stages in breast cancer (BC). Developing effective or preventive treatments is hampered by a lack of knowledge on the molecular mechanisms driving brain metastasis. Transmigration of BC cells through the brain endothelium is a key event in the pathogenesis of brain metastasis. In this study, we identified miR-101-3p as a critical micro-RNA able to reduce transmigration of BC cells through the brain endothelium. Our results revealed that miR-101-3p expression is downregulated in brain metastatic BC cells compared to less invasive variants, and varies inversely compared to the brain metastatic propensity of BC cells. Using a loss-and-gain of function approach, we found that miR-101-3p downregulation increased transmigration of BC cells through the brain endothelium in vitro by inducing COX-2 expression in cancer cells, whereas ectopic restoration of miR-101-3p exerted a metastasis-reducing effect. In regulatory experiments, we found that miR-101-3p mediated its effect by modulating COX-2-MMP1 signaling capable of degrading the inter-endothelial junctions (claudin-5 and VE-cadherin), key components of the brain endothelium. These findings suggest that miR-101-3p plays a critical role in the transmigration of breast cancer cells through the brain endothelium by modulating the COX-2-MMP1 signaling and thus may serve as a therapeutic target that can be exploited to prevent or suppress brain metastasis in human breast cancer.

Comparative metabolomics of MCF-7 breast cancer cells using different extraction solvents assessed by mass spectroscopy.

Metabolic profiling of cancer cells can play a vital role in revealing the molecular bases of cancer development and progression. In this study, gas chromatography coupled with mass spectrometry (GC-MS) was employed for the determination of signatures found in ER+/PR+ breast cancer cells derived from MCF-7 using different extraction solvents including: A, formic acid in water; B, ammonium hydroxide in water; C, ethyl acetate; D, methanol: water (1:1, v/v); and E, acetonitrile: water (1:1, v/v). The greatest extraction rate and diversity of metabolites occurs with extraction solvents A and E. Extraction solvent D showed moderate extraction efficiency, whereas extraction solvent B and C showed inferior metabolite diversity. Metabolite set enrichment analysis (MSEA) results showed energy production pathways to be key in MCF-7 cell lines. This study showed that mass spectrometry could identify key metabolites associated with cancers. The highest enriched pathways were related to energy production as well as Warburg effect pathways, which may shed light on how energy metabolism has been hijacked to encourage tumour progression and eventually metastasis in breast cancer.

Modulation of DNA damage response and induction of apoptosis mediates synergism between doxorubicin and a new imidazopyridine derivative in breast and lung cancer cells.

DNA damage response machinery (DDR) is an attractive target of cancer therapy. Modulation of DDR network may alter the response of cancer cells to DNA damaging anticancer drugs such as doxorubicin. The aim of the present study is to investigate the effects of a newly developed imidazopyridine (IAZP) derivative on the DDR after induction of DNA damage in cancer cells by doxorubicin. Cytotoxicity sulphrhodamine-B assay showed a weak anti-proliferative effect of IAZP alone on six cancer cell lines (MCF7, A549, A549DOX11, HepG2, HeLa and M8) and a normal fibroblast strain. Combination of IAZP with doxorubicin resulted in synergism in lung (A549) and breast (MCF7) cancer cells but neither in the other cancer cell lines nor in normal fibroblasts. Molecular studies revealed that synergism is mediated by modulation of DNA damage response and induction of apoptosis. Using constant-field gel electrophoresis and immunofluorescence detection of γ-H2AX foci, IAZP was shown to inhibit the repair of doxorubicin-induced DNA damage in A549 and MCF7 cells. Immunoblot analysis showed that IAZP suppresses the phosphorylation of the ataxia lelangiectasia and Rad3 related (ATR) protein, which is an important player in the response of cancer cells to chemotherapy-induced DNA damage. Moreover, IAZP augmented the doxorubicin-induced degradation of p21, activation of p53, CDK2, caspase 3/7 and phosphorylation of Rb protein. These effects enhanced doxorubicin-induced apoptosis in both cell lines. Our results indicate that IAZP is a promising agent that may enhance the cytotoxic effects of doxorubicin on some cancer cells through targeting the DDR. It is a preliminary step toward the clinical application of IAZP in combination with anticancer drugs and opens the avenue for the development of compounds targeting the DDR pathway that might improve the therapeutic index of anticancer drugs and enhance their cure rate.

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.

Distinct roles of XRCC4 and Ku80 in non-homologous end-joining of endonuclease- and ionizing radiation-induced DNA double-strand breaks.

Non-homologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) is mediated by two protein complexes comprising Ku80/Ku70/DNA-PKcs/Artemis and XRCC4/LigaseIV/XLF. Loss of Ku or XRCC4/LigaseIV function compromises the rejoining of radiation-induced DSBs and leads to defective V(D)J recombination. In this study, we sought to define how XRCC4 and Ku80 affect NHEJ of site-directed chromosomal DSBs in murine fibroblasts. We employed a recently developed reporter system based on the rejoining of I-SceI endonuclease-induced DSBs. We found that the frequency of NHEJ was reduced by more than 20-fold in XRCC4-/- compared to XRCC4+/+ cells, while a Ku80 knock-out reduced the rejoining efficiency by only 1.4-fold. In contrast, lack of either XRCC4 or Ku80 increased end degradation and shifted repair towards a mode that used longer terminal microhomologies for rejoining. However, both proteins proved to be essential for the repair of radiation-induced DSBs. The remarkably different phenotype of XRCC4- and Ku80-deficient cells with regard to the repair of enzyme-induced DSBs mirrors the embryonic lethality of XRCC4 knock-out mice as opposed to the viability of the Ku80 knock-out. Thus, I-SceI-induced breaks may resemble DSBs arising during normal DNA metabolism and mouse development. The removal of these breaks likely has different genetic requirements than the repair of radiation-induced DSBs.

Nonhomologous end-joining of site-specific but not of radiation-induced DNA double-strand breaks is reduced in the presence of wild-type p53.

Nonhomologous end-joining (NHEJ) of DNA double-strand breaks (DSBs) entails two principal mechanisms: modification of DNA ends prior to ligation (error-prone rejoining) or precise ligation without modification if the DNA ends are complementary (error-free repair). Error-prone rejoining is mutagenic, because it can lead to destruction of coding sequence or to chromosomal aberrations, and therefore must be tightly regulated. Previous studies on the role of the p53 tumor suppressor in the regulation of NHEJ have yielded conflicting results, but a rigorous analysis of NHEJ proficiency and fidelity in a purely chromosomal context has not been carried out. To this end, we created novel repair plasmid substrates that integrate into the genome. DSBs generated by the I-SceI endonuclease within these substrates were repaired by either error-prone rejoining or precise ligation. We found that the expression of wild-type p53 inhibited any repair-associated DNA sequence deletion, including a more than 250-fold inhibition of error-prone rejoining events compared to p53-null cells, while any promoting effect of p53 on precise ligation could not be directly evaluated. The role of p53 in NHEJ appeared to involve a direct transactivation-independent mechanism, possibly restricting DNA end-modification by blocking the annealing of single strands along flanking stretches of microhomology. The inhibition of error-prone rejoining by p53 did not apply to the rejoining of DSBs induced by ionizing radiation. In conclusion, our data suggest that p53 restricts the mutagenic effects of NHEJ without compromising repair proficiency or cell survival, thereby maintaining genomic stability.

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.