Cytotoxic Withanolide Constituents of Physalis longifolia

Fourteen new withanolides, 1–14, named withalongolides A–N, respectively, were isolated from the aerial parts of Physalis longifolia together with eight known compounds (15–22). The structures of compounds 1–14 were elucidated through spectroscopic techniques and chemical methods. In addition, the structures of withanolides 1, 2, 3, and 6 were confirmed by X-ray crystallographic analysis. Using a MTS viability assay, eight withanolides (1, 2, 3, 7, 8, 15, 16, and 19) and four acetylated derivatives (1a, 1b, 2a, and 2b) showed potent cytotoxicity against human head and neck squamous cell carcinoma (JMAR and MDA-1986), melanoma (B16F10 and SKMEL-28), and normal fetal fibroblast (MRC-5) cells with IC50 values in the range between 0.067 and 9.3 μM

EGFR Tyrosine Kinase Inhibitors Activate Autophagy as a Cytoprotective Response in Human Lung Cancer Cells

Epidermal growth factor receptor tyrosine kinase inhibitors gefitinib and erlotinib have been widely used in patients with non-small-cell lung cancer. Unfortunately, the efficacy of EGFR-TKIs is limited because of natural and acquired resistance. As a novel cytoprotective mechanism for tumor cell to survive under unfavorable conditions, autophagy has been proposed to play a role in drug resistance of tumor cells. Whether autophagy can be activated by gefitinib or erlotinib and thereby impair the sensitivity of targeted therapy to lung cancer cells remains unknown. Here, we first report that gefitinib or erlotinib can induce a high level of autophagy, which was accompanied by the inhibition of the PI3K/Akt/mTOR signaling pathway. Moreover, cytotoxicity induced by gefitinib or erlotinib was greatly enhanced after autophagy inhibition by the pharmacological inhibitor chloroquine (CQ) and siRNAs targeting ATG5 and ATG7, the most important components for the formation of autophagosome. Interestingly, EGFR-TKIs can still induce cell autophagy even after EGFR expression was reduced by EGFR specific siRNAs. In conclusion, we found that autophagy can be activated by EGFR-TKIs in lung cancer cells and inhibition of autophagy augmented the growth inhibitory effect of EGFR-TKIs. Autophagy inhibition thus represents a promising approach to improve the efficacy of EGFR-TKIs in the treatment of patients with advanced non-small-cell lung cancer

Novel internal regulators and candidate miRNAs within miR-379/miR-656 miRNA cluster can alter cellular phenotype of human glioblastoma

Clustered miRNAs can affect functioning of downstream pathways due to possible coordinated function. We observed 78-88% of the miR-379/miR-656 cluster (C14MC) miRNAs were downregulated in three sub-types of diffuse gliomas, which was also corroborated with analysis from The Cancer Genome Atlas (TCGA) datasets. The miRNA expression levels decreased with increasing tumor grade, indicating this downregulation as an early event in gliomagenesis. Higher expression of the C14MC miRNAs significantly improved glioblastioma prognosis (Pearson’s r=0.62; p<3.08e-22). ENCODE meta-data analysis, followed by reporter assays validated existence of two novel internal regulators within C14MC. CRISPR activation of the most efficient internal regulator specifically induced members of the downstream miRNA sub-cluster and apoptosis in glioblastoma cells. Luciferase assays validated novel targets for miR-134 and miR-485-5p, two miRNAs from C14MC with the most number of target genes relevant for glioma. Overexpression of miR-134 and miR-485-5p in human glioblastoma cells suppressed invasion and proliferation, respectively. Furthermore, apoptosis was induced by both miRs, individually and in combination. The results emphasize the tumor suppressive role of C14MC in diffuse gliomas, and identifies two specific miRNAs with potential therapeutic value and towards better disease management and therapy

Comparative Lipidomics in Clinical Isolates of Candida albicans Reveal Crosstalk between Mitochondria, Cell Wall Integrity and Azole Resistance

Prolonged usage of antifungal azoles which target enzymes involved in lipid biosynthesis invariably leads to the development of multi-drug resistance (MDR) in Candida albicans. We had earlier shown that membrane lipids and their fluidity are closely linked to the MDR phenomenon. In one of our recent studies involving comparative lipidomics between azole susceptible (AS) and azole resistant (AR) matched pair clinical isolates of C. albicans, we could not see consistent differences in the lipid profiles of AS and AR strains because they came from different patients and so in this study, we have used genetically related variant recovered from the same patient collected over a period of 2-years. During this time, the levels of fluconazole (FLC) resistance of the strain increased by over 200-fold. By comparing the lipid profiles of select isolates, we were able to observe gradual and statistically significant changes in several lipid classes, particularly in plasma membrane microdomain specific lipids such as mannosylinositolphosphorylceramides and ergosterol, and in a mitochondrial specific phosphoglyceride, phosphatidyl glycerol. Superimposed with these quantitative and qualitative changes in the lipid profiles, were simultaneous changes at the molecular lipid species levels which again coincided with the development of resistance to FLC. Reverse transcriptase-PCR of the key genes of the lipid metabolism validated lipidomic picture. Taken together, this study illustrates how the gradual corrective changes in Candida lipidome correspond to the development of FLC tolerance. Our study also shows a first instance of the mitochondrial membrane dysfunction and defective cell wall (CW) in clinical AR isolates of C. albicans, and provides evidence of a cross-talk between mitochondrial lipid homeostasis, CW integrity and azole tolerance

Role of genetic polymorphisms in tumour angiogenesis

Angiogenesis plays a crucial role in the development, growth and spread of solid tumours. Pro- and anti-angiogenic factors are abnormally expressed in tumours, influencing tumour angiogenesis, growth and progression. Polymorphisms in genes encoding angiogenic factors or their receptors may alter protein expression and/or activity. This article reviews the literature to determine the possible role of angiogenesis-related polymorphisms in cancer. Further research studies in this potentially crucial area of tumour biology are proposed

Suppression of Osteosarcoma Cell Invasion by Chemotherapy Is Mediated by Urokinase Plasminogen Activator Activity via Up-Regulation of EGR1

Background: The cellular and molecular mechanisms of tumour response following chemotherapy are largely unknown. We found that low dose anti-tumour agents up-regulate early growth response 1 (EGR1) expression. EGR1 is a member of the immediate-early gene group of transcription factors which modulate transcription of multiple genes involved in cell proliferation, differentiation, and development. It has been reported that EGR1 act as either tumour promoting factor or suppressor. We therefore examined the expression and function of EGR1 in osteosarcoma. Methods: We investigated the expression of EGR1 in human osteosarcoma cell lines and biopsy specimens. We next examined the expression of EGR1 following anti-tumour agents treatment. To examine the function of EGR1 in osteosarcoma, we assessed the tumour growth and invasion in vitro and in vivo. Results: Real-time PCR revealed that EGR1 was down-regulated both in osteosarcoma cell lines and osteosarcoma patients’ biopsy specimens. In addition, EGR1 was up-regulated both in osteosarcoma patient’ specimens and osteosarcoma cell lines following anti-tumour agent treatment. Although forced expression of EGR1 did not prevent osteosarcoma growth, forced expression of EGR1 prevented osteosarcoma cell invasion in vitro. In addition, forced expression of EGR1 promoted downregulation of urokinase plasminogen activator, urokinase receptor, and urokinase plasminogen activity. Xenograft mice models showed that forced expression of EGR1 prevents osteosarcoma cell migration into blood vessels. Conclusions: These findings suggest that although chemotherapy could not prevent osteosarcoma growth in chemotherapy-resistant patients, it did prevent osteosarcoma cell invasion by down-regulation of urokinase plasminogen activity via up-regulation of EGR1 during chemotherapy periods