Anemarrhena asphodeloides Bunge and its constituent timosaponin-AIII induce cell cycle arrest and apoptosis in pancreatic cancer cells.

Pancreatic cancer is one of the most recalcitrant and lethal of all cancers. We examined the effects of Anemarrhena asphodeloides (AA) and timosaponin-AIII (TAIII), a steroidal saponin present in AA, on pancreatic cancer cell proliferation and aimed to elucidate their potential apoptotic mechanisms of action. Viability assays and cell cycle analysis revealed that both AA and TAIII significantly inhibited pancreatic cancer cell proliferation and cell cycle progression compared to treatment with gemcitabine, the standard chemotherapeutic agent for advanced pancreatic cancer. We identified a dose-dependent increase in caspase-dependent apoptosis and activation of pro-apoptotic PI3K/Akt pathway proteins, with a subsequent downregulation of pro-survival PI3K/Akt pathway proteins, in pancreatic cancer cells treated with AA or TAIII over those treated with gemcitabine.

MIR494 reduces renal cancer cell survival coinciding with increased lipid droplets and mitochondrial changes.

BACKGROUND: miRNAs can regulate cellular survival in various cancer cell types. Recent evidence implicates the formation of lipid droplets as a hallmark event during apoptotic cell death response. It is presently unknown whether MIR494, located at 14q32 which is deleted in renal cancers, reduces cell survival in renal cancer cells and if this process is accompanied by changes in the number of lipid droplets. METHODS: 769-P renal carcinoma cells were utilized for this study. Control or MIR494 mimic was expressed in these cells following which cell viability (via crystal violet) and apoptotic cell numbers (via Annexin V/PI staining) were assessed. By western blotting, MIR494 cellular responses were validated using MIR494 antagomir and Argonaute 2 siRNA. Transmission electron microscopy (TEM) was performed in MIR494-transfected 769-P cells to identify ultrastructural changes. LipidTOX green neutral lipid staining and cholesterol measurements were conducted to assess accumulation of lipids droplets and total cholesterol levels, respectively, in MIR494 expressing 769-P cells. Indirect immunofluorescence and western analyses were also performed to examine changes in mitochondria organization. Co-transfection of MIR494 mimic with siRNA targeting LC3B and ATG7 was conducted to assess their contribution to formation of lipid droplets in MIR494-expressing cells. RESULTS: MIR494 expression reduces viability of 769-P renal cancer cells; this was accompanied by increased cleaved PARP (an apoptotic marker) and LC3B protein. Further, expression of MIR494 increased LC3B mRNA levels and LC3B promoter activity (2.01-fold; 50% increase). Interestingly, expression of MIR494 markedly increased multilamellar bodies and lipid droplets (by TEM and validated by LipidTOX immunostaining) while reducing total cholesterol levels. Via immunocytochemistry, we observed increased LC3B-associated endogenous punctae upon MIR494 expression. In contrast to ATG7 siRNA, knockdown of LC3B reduced the numbers of lipid droplets in MIR494-expressing cells. Our results also identified that MIR494 expression altered the organization of mitochondria which was accompanied by co-localization with LC3B punctae, decreased PINK1 protein, and altered Drp1 intracellular distribution. CONCLUSION: Collectively, our findings indicate that MIR494 reduces cell survival in 769-P renal cancer cells which is accompanied by increased lipid droplet formation (which occurs in a LC3B-dependent manner) and mitochondrial changes.

Roles and regulation of phospholipid scramblases.

Phospholipid scramblase activity is involved in the collapse of phospholipid (PL) asymmetry at the plasma membrane leading to externalization of phosphatidylserine. This activity is crucial for initiation of the blood coagulation cascade and for recognition/elimination of apoptotic cells by macrophages. Efforts to identify gene products associated with this activity led to the characterization of PL scramblase (PLSCR) and XKR family members which contribute to phosphatidylserine exposure in response to apoptotic stimuli. Meanwhile, TMEM16 family members were identified to externalize phosphatidylserine in response to elevated calcium in Scott syndrome platelets, which is critical for activation of the coagulation cascade. Herein, we report their mechanisms of gene regulation, molecular functions independent of their scrambling activity, and their potential roles in pathogenic conditions.