Cucurbitacin-B inhibits cancer cell migration by targeting mortalin and HDM2: computational and in vitro experimental evidence.

Cancer metastasis, a highly complex process wherein cancer cells move from the primary site to other sites in the body, is a major hurdle in its therapeutics. A large array of synthetic chemotherapeutic molecules used for the treatment of metastatic cancers, besides being extremely expensive and unaffordable, are known to cause severe adverse effects leading to poor quality of life (QOL) of the patients. In this premise, natural compounds (considered safe, easily available and economic) that possess the potential to inhibit migration of cancer cells are deemed useful and hence are on demand. Cucurbitacin-B (19-(10→9ß)-abeo-10-lanost-5-ene triterpene, called Cuc-B) is a steroid mostly found in plants of Cucurbitaceae family. It has been shown to possess anticancer activity although the molecular mechanism remains poorly defined. We present evidence that Cuc-B has the ability to interact with mortalin and HDM2 proteins that are enriched in cancer cells, suppress wild type p53 function and promote cancer cell migration. Computational analyses showed that Cuc-B interacts with mortalin similar to MKT077 and Withanone, both have been shown to reactivate p53 function and inhibit cell migration. Furthermore, Cuc-B interacted with HDM2 similar to Y30, a well-known inhibitor of HDM2. Experimental cell and molecular analyses demonstrated the downregulation of several proteins, critically involved in cell migration in Cuc-B (low non-toxic doses)-treated cancer cells and exhibited inhibition of cell migration. The data suggested that Cuc-B is a potential natural drug that warrants further mechanistic and clinical studies for its use in the management of metastatic cancers.Communicated by Ramaswamy H. Sarma.

Functional characterization of miR-708 microRNA in telomerase positive and negative human cancer cells.

Activation of a telomere length maintenance mechanism (TMM), including telomerase and alternative lengthening of telomeres (ALT), is essential for replicative immortality of tumor cells, although its regulatory mechanisms are incompletely understood. We conducted a microRNA (miRNA) microarray analysis on isogenic telomerase positive (TEP) and ALT cancer cell lines. Amongst nine miRNAs that showed difference in their expression in TEP and ALT cancer cells in array analysis, miR-708 was selected for further analysis since it was consistently highly expressed in a large panel of ALT cells. miR-708 in TEP and ALT cancer cells was not correlated with C-circle levels, an established feature of ALT cells. Its overexpression induced suppression of cell migration, invasion, and angiogenesis in both TEP and ALT cells, although cell proliferation was inhibited only in TEP cells suggesting that ALT cells may have acquired the ability to escape inhibition of cell proliferation by sustained miR-708 overexpression. Further, cell proliferation regulation in TEP cells by miR708 appears to be through the CARF-p53 pathway. We demonstrate here that miR-708 (i) is the first miRNA shown to be differentially regulated in TEP and ALT cancer cells, (ii) possesses tumor suppressor function, and (iii) deregulates CARF and p21WAF1-mediated signaling to limit proliferation in TEP cells.

Induction of Senescence in Cancer Cells by a Novel Combination of Cucurbitacin B and Withanone: Molecular Mechanism and Therapeutic Potential.

Cancer, an uncontrolled proliferation syndrome, is treated with synthetic chemotherapeutic drugs that are associated with severe adverse effects. Development and application of new natural compounds is warranted to deal with the exponentially increasing incidence of cancer worldwide. Keeping selective toxicity to cancer cells as a priority criterion, we developed a combination of Cucurbitacin B and Withanone, and analyzed its anticancer potential using non-small cell lung cancer cells. We demonstrate that the selective cytotoxicity of the combination, called CucWi-N, to cancer cells is mediated by induction of cellular senescence that was characterized by decrease in Lamin A/C, CDK2, CDK4, Cyclin D, Cyclin E, phosphorylated RB, mortalin and increase in p53 and CARF proteins. It compromised cancer cell migration that was mediated by decrease in mortalin, hnRNP-K, vascular endothelial growth factor, matrix metalloproteinase 2, and fibronectin. We provide in silico, molecular dynamics and experimental data to support that CucWi-N (i) possesses high capability to target mortalin-p53 interaction and hnRNP-K proteins, (ii) triggers replicative senescence and inhibits metastatic potential of the cancer cells, and (iii) inhibits tumor progression and metastasis in vivo. We propose that CucWi-N is a potential natural anticancer drug that warrants further mechanistic and clinical studies.

Integration of conventional cell viability assays for reliable and reproducible read-outs: experimental evidence.

OBJECTIVE: Short-term viability assays of cultured cells in 96-well plates are routinely used to determine the cytotoxicity or safety of drugs. These are often based on the formation of chromogen, generated selectively in viable cells. The innate problems of such short-term cell viability assays include (i) effect of drugs is determined by cell density (ii) some drugs have slow/gradual effect and hence may escape such assays, (iii) cell morphology that reveal significant hints to molecular signaling underlining the effect of drugs cannot be effectively captured, (iv) long-term effect on viability and clonogenic potential of cells cannot be determined and (v) herbal extracts often possess intrinsic color that interferes with spectrophotometer estimation. In light of the ease and importance of cell culture-based assessment of drug safety and cytotoxicity, we attempted to combine the conventional cell-based assays in a way that allows multiple readouts (quantitative and qualitative) from a single experiment, and avoids the drawbacks of color interference. RESULTS: We have established and validated (using 16 types of cultured mammalian cells) a Quantitative and Qualitative Cell Viability assay in 12-well cell culture plates. It overcomes several shortcomings as discussed above and allows long-term observations on cell morphology and clonogenicity.