Endophytic fungi of Panax sokpayensis produce bioactive ginsenoside Compound K in flask fermentation.

Endophytes of Panax have the potential to produce their host plant secondary metabolites, ginsenosides. Panax sokpayensis, an endemic traditional medicinal plant of the Sikkim Himalayas was explored for the isolation of endophytic fungi. In the present study, we have isolated 35 endophytic fungal cultures from the rhizome of P. sokpayensis and screened for ginsenosides production by HPLC by comparing the peak retention time with that of standard ginsenosides. The HPLC analysis revealed that out of 35 isolates, the mycelial extracts of four fungal endophytes (PSRF52, PSRF53, PSRF49 and PSRF58) exhibited peaks with a similar retention time of the standard ginsenoside, Compound K (CK). LC-ESI-MS/MS analysis led to the confirmation of ginsenoside CK production by the four fungal endophytes which showed a compound with m/z 639.6278, similar to that of standard ginsenoside CK with yield in potato dextrose broth flask fermentation ranging from 0.0019 to 0.0386 mg/g of mycelial mass in dry weight basis. The four prospective fungal endophyte isolates were identified as Thermothielavioides terrestris PSRF52, Aspergillus sp. PSRF49, Rutstroemiaceae sp. strain PSRF53, and Phaeosphaeriaceae sp. strain PSRF58 based on ITS sequencing. The present finding highlights the need for further study on growth optimization and other culture parameters to exploit the endophytes as an alternative source for ginsenoside CK production.

Combination of tamoxifen and D-limonene enhances therapeutic efficacy in breast cancer cells.

Breast cancer one of the most common diseases in women, has a high death and morbidity rate. Tamoxifen being very much effective in the chemoprevention of breast cancer has been shown to develop resistance during the course of treatment making it difficult for patient's survival. By combining tamoxifen with naturally occurring substances having similar activities, might control the toxicity and increase the susceptibility towards the treatment. As a natural compound, D-limonene has been reported to inhibit the growth of certain malignancies significantly. The main goal of our work is to investigate the combinatorial antitumor effects of D-limonene and tamoxifen in MCF-7 cells, as well as understand the potential underlying anticancer mechanism. MTT assays, colony formation assays, DAPI and Annexin V-FITC labeling, flow cytometer analysis, and western blot analysis were used to explore the details of anticancer mechanism. The combined effects of tamoxifen with D-limonene have shown significant decrease in the cell viability of MCF 7 cells. According to flow cytometer analyses and Annexin V/PI staining, D-limonene has been found to increase tamoxifen-mediated apoptosis as compared to the treatment alone in these cells. Additionally, cell growth has been found to be arrested at G1 phase by regulating cyclin D1 and cyclin B1. Our research consequently provided the first evidence that combining D-limonene and tamoxifen might increase the anticancer efficacy by inducing apoptosis in MCF 7 breast cancer cells. This combinatorial treatment strategy demands more research which might fulfill the need for improved treatment efficacy in controlling breast cancer.

Proximal discrepancy in intrinsic atomic interaction arrests G2/M phase by inhibiting Cyclin B1/CDK1 to infer molecular and cellular biocompatibility of D-limonene.

The quest for different natural compounds for different biomedical applications especially in the treatment of cancer is at a high pace with increasing incidence of severity. D-limonene has been portrayed as one of the effective potential candidate centered to the context of breast cancer. The anticipation of its count as an effective biomedical agent required a detailed understanding of their molecular mechanism of biocompatibility. This study elucidates the mechanistic action of D-limonene channelized by the induction of apoptosis for controlling proliferation in breast cancer cells. The possible mechanism was explored through an experimental and computational approach to estimate cell proliferation inhibition, cell cycle phase distribution, apoptosis analysis using a flow cytometry, western blotting and molecular docking. The results showed reduced dose and time-dependent viability of MCF7 cells. The study suggested the arrest of the cell cycle at G2/M phase leading to apoptosis and other discrepancies of molecular activity mediated via significant alteration in protein expression pattern of anti-apoptotic proteins like Cyclin B1 and CDK1. Computational analysis showed firm interaction of D-limonene with Cyclin B1 and CDK1 proteins influencing their structural and functional integrity indicating the mediation of mechanism. This study concluded that D-limonene suppresses the proliferation of breast cancer cells by inducing G2/M phase arrest via deregulation of Cyclin B1/CDK1.

Molecular genetics analysis of hereditary breast and ovarian cancer patients in India.

BACKGROUND: Hereditary cancers account for 5-10% of cancers. In this study BRCA1, BRCA2 and CHEK2*(1100delC) were analyzed for mutations in 91 HBOC/HBC/HOC families and early onset breast and early onset ovarian cancer cases. METHODS: PCR-DHPLC was used for mutation screening followed by DNA sequencing for identification and confirmation of mutations. Kaplan-Meier survival probabilities were computed for five-year survival data on Breast and Ovarian cancer cases separately, and differences were tested using the Log-rank test. RESULTS: Fifteen (16%) pathogenic mutations (12 in BRCA1 and 3 in BRCA2), of which six were novel BRCA1 mutations were identified. None of the cases showed CHEK2*1100delC mutation. Many reported polymorphisms in the exonic and intronic regions of BRCA1 and BRCA2 were also seen. The mutation status and the polymorphisms were analyzed for association with the clinico-pathological features like age, stage, grade, histology, disease status, survival (overall and disease free) and with prognostic molecular markers (ER, PR, c-erbB2 and p53). CONCLUSION: The stage of the disease at diagnosis was the only statistically significant (p < 0.0035) prognostic parameter. The mutation frequency and the polymorphisms were similar to reports on other ethnic populations. The lack of association between the clinico-pathological variables, mutation status and the disease status is likely to be due to the small numbers.

Involvement of YY1 and its correlation with c-myc in NDEA induced hepatocarcinogenesis, its prevention by d-limonene.

Anticarcinogenic activity of d-limonene has been well documented within last few years. We have also reported the anticarcinogenic activity of d-limonene in N-nitrosodiethylamine (NDEA) induced hepatocarcinogenesis. The involvement of oncogenes which adds to the mechanisms of d-limonene mediated chemprevention were also suggested in the same model system. The overexpression of c-myc oncoprotein in different durations of NDEA induced hepatrocarcinogenesis is observed which is inhibited completely when d-limonene was treated prior to and along with NDEA. To work further in this direction, an attempt has been made here to know the role of YY1 (Yin Yang 1) transcription factor in N-nitrodiethylamine (NDEA) induced hepatocarcinogenesis and its chemoprevention by d-limonene. Electrophoretic mobility shift assay results have clearly indicated the binding of YY1 in control liver tissue. But this binding is blocked in 60 days and 150 days NDEA treated liver tumors. Thus, it is assumed that there is deregulation of YY1 transcription factor in NDEA induced hepatocarcinogenesis. A similar type of binding to that of control liver tissue has also observed when limonene was given prior to NDEA administration. Western blot analysis has shown inhibition of YY1 protein in NDEA induced liver tumor samples in comparison to normal and both NDEA and limonene treated samples. On the otherhand RT-PCR analysis does not indicate any correlation between YY1 mRNA level and inhibition of YY1 protein. However, along with our earlier information about c-myc with the present study, clearly indicated the involvement of YY1 in NDEA induced hepatocarcinogenesis and d-limonene mediated chemoprevention which might be regulated by c-myc oncoprotein.

Rearrangement of p53 gene with overexpressed p53 protein in primary cervical cancer.

The frequency of p53 mutations is low and there is evidence of p53 protein overexpression even without p53 mutations in cervical cancers. This suggests that alternative mechanisms other than p53 mutation could be responsible for tumourigenesis of the uterine cervix. Therefore, an attempt has been made in the present investigation to analyze mutation and rearrangement of p53 gene in primary cervical cancers. The results indicated absence of mutation and presence of rearrangement in about 35% of cervical cancer patients. However, p53 overexpression in 50% of patients was demonstrated by immunohistochemistry and Western blot analysis. Further, rearrangement of p53 has been correlated with p53 mRNA and p53 protein status. The results indicated presence of overexpressed p53 protein in the samples with rearranged p53 gene. Thus, it is presumed that rearrangement of p53 might lead to production of defective p53 protein by affecting the level of p53 protein and this might have a role in the process of tumourigenesis. This study reports for the first time rearrangement of p53 in cervical cancers.