TL stimulation exhibited blunt activities of Complex I and II in p53-deficient HCT116 cells.

<p>(A-D) Specific enzymatic activities of ETC complex I, II, IV and of citrate synthase (CS) were measured cells lysates from TL (50 nM) stimulated or control HCT116 P53+/+ and HCT116p53-/- cells. (E, F) Representative Immunoblots for hNDUFA9 (complexes I), hSDHA (Complex II), and citrate synthase are presented (mean±SD; *p<0.05; n = 3).</p

Minnelide/Triptolide Impairs Mitochondrial Function by Regulating SIRT3 in P53-Dependent Manner in Non-Small Cell Lung Cancer

<div><p>Minnelide/Triptolide (TL) has recently emerged as a potent anticancer drug in non-small cell lung cancer (NSCLC). However, the precise mechanism of its action remains ambiguous. In this study, we elucidated the molecular basis for TL-induced cell death in context to p53 status. Cell death was attributed to dysfunction of mitochondrial bioenergetics in p53-deficient cells, which was characterized by decreased mitochondrial respiration, steady-state ATP level and membrane potential, but augmented reactive oxygen species (ROS). Increased ROS production resulted in oxidative stress in TL-treated cells. This was exhibited by elevated nuclear levels of a redox-sensitive transcriptional factor, NF-E2-related factor-2 (NRF2), along with diminished cellular glutathione (GSH) content. We further demonstrated that in the absence of p53, TL blunted the expression of mitochondrial SIRT3 triggering increased acetylation of NDUAF9 and succinate dehydrogenase, components of complexes I and II of the electron transport chain (ETC). TL-mediated hyperacetylation of complexes I and II proteins and these complexes displayed decreased enzymatic activities. We also provide the evidence that P53 regulate steady-state level of SIRT3 through Proteasome-Pathway. Finally, forced overexpression of Sirt3, but not deacetylase-deficient mutant of Sirt3 (H243Y), restored the deleterious effect of TL on p53-deficient cells by rescuing mitochondrial bioenergetics. On contrary, Sirt3 deficiency in the background of wild-type p53 triggered TL-induced mitochondrial impairment that echoed TL effect in p53-deficeint cells. These findings illustrate a novel mechanism by which TL exerts its potent effects on mitochondrial function and ultimately the viability of NSCLC tumor.</p></div

Silencing of Sirt3 sensitizes HCT116 53+/+ cells for TL-mediated mitochondrial dysfunction.

<p>(A-B) HCT116 p53+/+ cells were transfected scrambled siRNA (NS) or Sirt3 siRNA. After 48 hours, cells were treated with 50 nM Triptolide tor additional 6 hours before subjected for Seahorse XF analyzer for OCR. Basal (A) and Maximal (B) respiration were compared among the groups. (C-F) Cells from above (A,B) were also analyzed for (C) ATP level, (D) MitoSOX for mtROS, (E) Mitochondrial potential and (F) cell viability. (G) Cells from above were also subjected for cell viability assay. The data were expressed relative over NS-siRNA transfected cell treated with DMSO (mean±SD; *p<0.05, **p<0.001; n = 3)</p

Human P53 associates with M0069tochondrial Sirt3 and modulates its level through Proteasome Pathway.

<p>(A, B) p53 regulates Sirt3 level. Sirt3 expression at RNA (A) and protein level (B) were measured in p53 wild type (A549 and HCT116 p53+/+) and p53 deficient cells (H1299 and HCT116 p53-/-). Data are presented relative Sirt3 mRNA in p53 containing cells. (C, D) P53 interacts with SIRT3 in-vivo. HCT116 p53-/- cells were transfected with FLAG-p53 and Myc-Sirt3 alone or in combination. Twenty-four hours post-transfection, cells were harvested in immunoprecipitation assay buffer (IP Buffer). Cell lysates were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-Myc. Five percent of whole-cell extract (WCE) was used as an input and was directly immunoblotted with respective antibodies. (D) Interaction of endogenous P53 and SIRT3. HCT116 p53+/+ cells were stimulated with Triptolide (50 nM) for 6 hours. Cells were harvested in IP buffer and cell lysates were immunopreipiatated with P53 (DO1) antibody followed by immunoblotting with SIRT3 antibody. Five percent WCE was directly immunoblotted with SIRT3 and P53 antibody. (E) SIRT3 turnover in p53+/+ and p53-/- HCT116 cells. Cells were incubated with 50 μg/ml of Cycloheximide for the indicated time. Cell lysates were subjected for immunoblotting with SIRT3 (above panel), p53 (Middle Panel) and β-actin (lower panel). (F,G) SIRT3 degradation is through Proteasome Pathway involving SKP2 E3-ligase. HCT116 p53-/- cells were pretreated with MG132 (2 μM) for 1 hour and then were treated with Cycloheximide (50 μg/ml) for indicated time. SIRT3 and β-actin levels were detected by immunoblotting with respective antibodies. Indicated cell lysates were immunoblotted with anti-SKP2 to examine the expression of SKP2 level (G). (H) TL alters SKP2 levels in p53-/- cells. HCT116 (p53+/+ and p53-/-) cells were stimulated with indicated dose of TL for 6 hours. Cell lsyates were immunoblotted with anti-SKP2, anti-SIRT3 and anti-beta Actin antibodies.</p

TL induces hyperacetylation of ETC complexes through mitochondrial Sirt3.

<p>(A,B) P53 containing and deficient HCT116 cells were treated with TL (50 nM for 6 hours). Immunoprecipitates of anti-acetylayed lysine were immunoblotted with hNDUFA9 (A, top panel) or succinate dehydrogenase (hSDHA) (B, top panel). Whole-cell lysates were immunoblotted with hNDUFA9 (A, lower panel) and SDHA (B, lower Panel) for the input. (C) TL down-regulates SIRT3 expression. Whole-cell lysates from HCT116 cells (p53 +/+ and p53-/-) induced by DMSO or TL were immunoblotted with anti-SIRT3, P53, and beta-actin (top panel). Relative quantity of SIRT3 was computed after normalization with beta-actin (mean±SD; *p<0.05; n = 3).</p

TL induces oxidative stress in p53-Deficient HCT116 cells.

<p>(A,B,C) TL treatment led to nuclear accumulation of NRF2 and its target genes (HO1 and NQO1). (A) HCT116 p53+/+ and p53-/- cells were treated with TL (50nM for 30 and 60 minutes). Nuclear extracts were prepared and subjected to immunoblotting with NRF2, NQO1 and HO1. Representative Immunoblots are presented. (B,C,D) Relative protein quantity normalized to PCNA is presented (**p<0.01, n = 3). (E) TL decreases glutathione content. The glutathione levels were measured in cell lysates from TL-induced and control HCT116p53+/+ and p53-/- cells. The levels were normalized with protein content and the data was represented as relative content of %control (*p<0.05). (F-H) TL inhibits SOD2 levels and activity in p53-depleted cells. (F,G) Immunoblotting for Mn-SOD (SOD2) was performed in cell lysates from HCT116 p53+/+ and p53-/- stimulated with DMSO or TL. Relative SOD2 protein levels normalized to beta-actin. (H) SOD activity was measured in cell lysates from F. The data was presented as %activity (mean±SD; *p<0.05; n = 3).</p

Mitochondrial SIRT3 mitigates TL-induced mitochondrial dysfunction in p53-deficient cells.

<p>(A-D) SIRT3 overexpression rescues mitochondrial respiration in TL-stimulated HCT116 p53-/- cells. P53 deficient HCT116 cells were transfected with vector, myc-SIRT3 or myc-SIRT3 (H243Y). 24 hours of post-transfection, cells were stimulated with TL (50 nM) for 6 hours and were subjected for analyzing oxygen consumption rate (OCR) using the Seahorse Bioscience Extra Cellular Flux analyzer. Data were normalized with protein content and presented as basal (A), non-ATP-linked (B), maximal (C) and non-mitochondrial respiration (D) (mean±SEM; *p<0.05; n = 3). (E-G) SIRT3, but not deacetylase deficient mutant of SIRT3, abrogates TL-induced alteration in ROS production, membrane potential and ATP level. HCT116 p53-/- cells were transiently transfected with indicated plasmids. After 24 hours of transfection, cells were stimulated with TL (50 nM) for additional 6 hours. Mitochondrial ROS (mtROS) (E), ATP level (F) and mitochondrial membrane potential (G) were measured. Relative values were presented as fold change over control ((mean±SD; **p<0.001; n = 3). (H) Cells transfected with Myc-SIRT3 or Myc-SIRT3 (H243Y) were stimulated with 50 nM of TL and cell viability was assesses using CyQuont cell proliferation assay kit. The data was presented as % viable cells over vector transfected DMSO stimulated cells. (I) Protein lysates from vector, Myc-SIRT3 or Myc-SIRT3 (H243Y) transfected HCT116 p53-/- cells were immunoblotted with Myc antisera to assess the expression of overexpressed SIRT3 or SIRT3 (H243Y) mutant.</p

Minnelide: A Novel Therapeutic That Promotes Apoptosis in Non-Small Cell Lung Carcinoma In Vivo

<div><p>Background</p><p>Minnelide, a pro-drug of triptolide, has recently emerged as a potent anticancer agent. The precise mechanisms of its cytotoxic effects remain unclear.</p> <p>Methods</p><p>Cell viability was studied using CCK8 assay. Cell proliferation was measured real-time on cultured cells using Electric Cell Substrate Impedence Sensing (ECIS). Apoptosis was assayed by Caspase activity on cultured lung cancer cells and TUNEL staining on tissue sections. Expression of pro-survival and anti-apoptotic genes (<i>HSP70</i>, <i>BIRC5, BIRC4, BIRC2, UACA, APAF-1</i>) was estimated by qRTPCR. Effect of Minnelide on proliferative cells in the tissue was estimated by Ki-67 staining of animal tissue sections.</p> <p>Results</p><p>In this study, we investigated <i>in</i><i>vitro</i> and <i>in</i><i>vivo</i> antitumor effects of triptolide/Minnelide in non-small cell lung carcinoma (NSCLC). Triptolide/Minnelide exhibited anti-proliferative effects and induced apoptosis in NSCLC cell lines and NSCLC mouse models. Triptolide/Minnelide significantly down-regulated the expression of pro-survival and anti-apoptotic genes (<i>HSP70</i>, <i>BIRC5, BIRC4, BIRC2, UACA</i>) and up-regulated pro-apoptotic <i>APAF-1</i> gene, in part, via attenuating the NF-κB signaling activity.</p> <p>Conclusion</p><p>In conclusion, our results provide supporting mechanistic evidence for Minnelide as a potential in NSCLC.</p> </div

Minnelide leads to tumor regression in xenograft mouse models.

<p>In two xenograft mouse models, A549 (right panel) and NCI-H460 (left panel), tumor volumes were compared between minnelide treated (n = 10) and untreated groups (n = 10). Five days after tumor injection, mice began receiving daily intraperitoneal injections of minnelide at 0.42 mg/kg mouse weight. Control animals were injected with equivalent volumes of phosphate-buffered saline. Suppression of tumor growth occurred in the minnelide treated groups in comparison with the control groups in both xenograft models (A, E). Graphs (B and F) showed significantly decreased tumor volume from animals (A and E). Results were normalized to the untreated group for each cell line and expressed as the mean, <i>Columns</i>, <i>bars</i>, SE. Ki-67 protein expression was significantly decreased in the tumor tissue of minnelide treated group in both mouse models compare to saline treated groups (C and G). In concordance with decreased Ki-67 staining, TUNEL staining shown increased number of apoptotic cells in both mouse models (D and H). <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p

Triptolide decreases proliferation and viability of NSCLC cells.

<p>A549 cells (right panel) and NCI-H460 cells (left panel) were treated with 25-200 nM of triptolide for times indicated. Proliferation (A, E) and viability (B, F), as well as BrdU incorporation (C, G) were significantly reduced of both cell lines were reduced; however, the cytotoxic effect of triptolide was more pronounced in NCI-H460 cells. The indicator of DNA repair, phosphorylated H2AX, was significantly increased in both cell lines with triptolide treatment as shown (D, H). <i>Columns</i>, mean, <i>bars</i>, SE. Statistical significance of results was calculated with the Student`s <i>t</i> test (N=3) *<i>P</i> = 0.05; **<i>P</i> = 0.005.</p