Interaction of C20-substituted derivative of pregnenolone acetate with copper (II) leads to ROS generation, DNA cleavage and apoptosis in cervical cancer cells: Therapeutic potential of copper chelation for cancer treatment.

Cervical cancer is a leading cause of cancer-related deaths among women in developing countries. Therefore, development of new chemotherapeutic agents is required. Unlike normal cells, cancer cells contain elevated copper levels which play an integral role in angiogenesis. Thus, targeting copper via copper-specific chelators in cancer cells can serve as effective anticancer strategy. In this work, a copper chelator pregnenolone acetate nucleus-based tetrazole derivative (ligand-L) was synthesized and characterized by elemental analysis, ESI-MS, 1H NMR and 13C NMR. DNA binding ability of ligand-L was studied using UV-Vis and fluorescence spectroscopy. Fluorescence spectroscopy studies reveal that quenching constant of ligand-l-DNA and ligand-L-Cu(II) were found to be 7.4 × 103 M-1 and 8.8 × 103 M-1, respectively. In vitro toxicity of ligand-L was studied on human cervical cancer C33A cancer cells. Results showed that ligand-L exhibit significant cytotoxic activity against cervical cancer C33A cells with IC50 value 5.0 ±â€¯1.8 µM. Further, it was found that ligand-L cytotoxicity is due to redox cycling of copper to generate ROS which leads to DNA damage and apoptosis. In conclusion, this is the report where we synthesized pregnenolone acetate-based tetrazole derivative against C33A cells that targets cellular copper to induce pro-oxidant death in cancer cells. These findings will provide significant insights into the development of new chemical molecules with better copper chelating and pro-oxidant properties against cancer cells.

Deciphering the molecular mechanism underlying anticancer activity of coumestrol in triple-negative breast cancer cells.

Triple-negative breast cancer (TNBC) represents the highly aggressive subgroup of breast cancers with poor prognosis due to absence of estrogen receptor (ER). Therefore, alternative targeted therapies are required against ER-negative breast cancers. Coumestrol, a phytoestrogen inhibits cell growth of ER-negative breast cancer MDA-MB-231 cells; the exact mechanism has not yet been reported. Unlike normal cells, cancer cells contain elevated copper which play an integral role in angiogenesis. The current focus of the work was to identify any possible role of copper in coumestrol cytotoxic action against breast cancer MDA-MB-231 cells. Results demonstrated that coumestrol inhibited cell viability, induced ROS generation, DNA damage, G1/S cell cycle arrest, up-regulation of Bax and apoptosis induction via caspase-dependent mitochondrial mediated pathway in MDA-MB-231 cells. Further, addition of copper chelator, neocuproine and ROS scavenger, N-acetyl cysteine were ineffective in abrogating coumestrol-mediated apoptosis. This suggests non-involvement of copper and ROS in coumestrol-induced apoptosis. To account for coumestrol-mediated up-regulation of Bax and apoptosis induction, direct binding potential between coumestrol and Bax/Bcl-2 was studied using in silico molecular docking studies. We propose that coumestrol directly enters cells and combines with Bax/Bcl-2 to alter their structures, thereby causing Bax binding to the outer mitochondrial membrane and Bcl-2 release from the mitochondria to initiate apoptosis. Thus, non-copper targeted ROS independent DNA damage is the central mechanism of coumestrol in ER-negative MDA-MB-231 cells. These findings will be useful in better understanding of anticancer mechanisms of coumestrol and establishing it as a lead molecule for TNBC treatment.

Towards therapeutic advances in melanoma management: An overview.

Melanoma is one of the most aggressive types of skin cancer with rapidly increasing incidence rate. The disease is largely considered incurable and the patients diagnosed with metastatic melanoma have a survival of not more than five years. Despite of the recent advances in anti-melanoma chemo- and immunotherapies, the available drugs are relatively toxic and responsive to only a limited subset of lesions. Currently, topical pharmacotherapy is demonstrated as an effective approach for the treatment of various skin cancers. Also, in vitro testing of melanoma cell lines and murine melanoma models has identified a number of relatively safe and effective phytochemicals. In this review, we described the use of topical pharmacotherapy for the treatment of skin cancers. Melanoma treatment by drugs targeting MAPK-pathway has also been discussed. Long non-coding RNAs and therapeutics targeting ER-associated pathways looks quite promising for the treatment of melanoma. Moreover, some natural anticancer compounds that have been reported to have anti-melanoma effects have also been described. At present a better understanding of genetics and epigenetics of initiation and progression of melanoma is needed for the identification of novel biomarkers and development of targeted therapeutics against melanoma.

Cytotoxic activity of soy phytoestrogen coumestrol against human breast cancer MCF-7 cells: Insights into the molecular mechanism.

Coumestrol is a phytoestrogen present in soybean products and recognized as potential cancer therapeutic agent against breast cancer. However, the clear molecular mechanism of anticancer-activity of coumestrol in breast carcinoma has not been reported. It is well established that copper levels are elevated in different malignancies. Therefore, the objective of this study was to investigate the copper-dependent cytotoxic action of coumestrol in human breast cancer MCF-7 cells. Results showed that coumestrol inhibited proliferation and induced apoptosis in MCF-7 cells, which was prevented by copper chelator neocuproine and ROS scavengers. Coumestrol treatment induced ROS generation coupled to DNA fragmentation, up-regulation of p53/p21, cell cycle arrest at G1/S phase, mitochondrial membrane depolarization and caspases 9/3 activation. All these effects were suppressed by ROS scavengers and neocuproine. These results suggest that coumestrol targets elevated copper for redox cycling to generate ROS leading to DNA fragmentation. DNA damage leads to p53 up-regulation which directs the cell cycle arrest at G1/S phase and promotes caspase-dependent apoptosis of MCF-7 cells. In conclusion, copper targeted ROS-mediated p53-dependent mechanism better explains the cytotoxic action of coumestrol in MCF-7 cells. Thus, targeting elevated copper levels might be a potential therapeutic strategy for selective cytotoxic action against malignant cells.

Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells.

Targeting the testis-specific heat-shock protein 70-2 (HSP70-2) reduces cellular growth, migration, and invasion in renal cell carcinoma cells.

Renal cell carcinoma (RCC) represents one of the most resistant tumors to radiotherapy and chemotherapy. Current therapies for the RCC patients are limited owing to lack of diagnosis and therapeutic treatments. Testis-specific heat-shock protein 70-2 (HSP70-2), a member of HSP70 chaperone family, has been shown to be associated with various cancers. In the present study, we investigated the putative role of HSP70-2 in various malignant properties of the RCC cells. HSP70-2 messenger RNA (mRNA) and protein expression was investigated in A704, ACHN, and Caki-1 cells derived from the RCC patients. We assessed the expression of HSP70-2 gene and protein by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting, respectively. The expression of HSP70-2 protein was further validated by performing indirect immunofluorescence (IIF) and flow cytometry. The malignant properties of high-grade invasive A704 and Caki-1 cells, such as cellular proliferation, colony formation, migration, invasion, and wound healing, were evaluated by silencing the expression of HSP70-2 gene in these cells. Statistical significance was defined using Student's t test. Our RT-PCR and Western blotting data showed the expression of HSP70-2 in all RCC cells. Our results showed that HSP70-2 was predominantly expressed in cytoplasm and found to be colocalized with endoplasmic reticulum, mitochondria, Golgi body, and plasma membrane but not the nuclear envelope. Knockdown of HSP70-2 expression with specific short hairpin RNA (shRNA) demonstrated significant reduction in cell growth and colony formation. Further, a marked reduction in cell migration and invasion was also observed, indicating the potential role of HSP70-2 in metastasis. Collectively, our data suggest that HSP70-2 plays a key role in cancerous growth and invasive potential of RCC cells. Thus, HSP70-2 could serve as a novel potential therapeutic target for the RCC.

Cancer testis antigens: A new paradigm for cancer therapy.

Cancer immunotherapy is a promising field with limited success, also due to lack of tumor-specific targets. In our attempt of exploring novel biomarkers and immunotherapeutic targets against cancer, we have discovered a novel cancer testis antigen, SPAG9, in cancers of different histological origin and demonstrated its potential role in oncogenesis.