Display Settings:

Items per page
We are sorry, but NCBI web applications do not support your browser and may not function properly. More information

Results: 6

1.
Figure 6

Figure 6. Model depicting mechanism of necroptosis induction by mTOR and autophagy inhibition.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

Renal carcinoma cells have activated mTOR, Nrf2 antioxidant defense and autophagy under basal conditions sufficient to manage ROS and survive. Autophagy inhibition with CQ blocks clearance of damaged mitochondrial through mitophagy. ROS is mitigated through the Nrf2 antioxidant defense pathway. mTOR inhibition promotes respiration and inhibits Nrf2 antioxidant defense. RIPKs are degraded by the ubiquitin proteasome system and damaged mitochondrial are cleared by mitophagy to maintain ROS homeostasis and cell survival. Pharmacological inhibition of both autophagy with CQ and mTOR with CCI-779 causes loss of antioxidant defense via Nrf2 and persistence of RIPKs and mitochondria leading to RIPK activation and ROS-mediated cell death by necroptosis.

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.
2.
Figure 3

Figure 3. Cell death induced by autophagy and mTOR inhibition is RIPK-dependent necroptosis.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

(A) Clonogenic survival assay showing Nec1 pretreatment rescues clonogenic survival. RCC cells were pretreated for one hour with Necrostatin1 (Nec1) followed by the addition of CQ, CCI-779 or combination of CQ and CCI-779 to the media. After 18 hours media was changed to normal culture media and cells were allowed to recover for 5 days. In 4 of 7 RCC cell lines (RCC4, A498, ACHN, 786O), Nec1 pretreatment rescued clonogenic survival from CQ and CCI. (B) Viability graph showing that siRNA knockdown of RIPK3 in RCC4 prevented cell death induced by an 18 hour drug treatment of combination of CQ and CCI-779 as compared to siRNA control siLamin (*p = 0.007). Error bars represent ± S.D. (C) Western blots of RNA knockdown of RIPK3. RCC4 cells were transfected with either Lamin- or RIPK3-siRNA and analyzed for levels of RIPK3 over 2 days. (D) Clonogenic survival assay showing Nec1 pretreatment or knockdown of essential necroptosis protein RIPK3 enhanced survival in the combination of CQ and CCI-779 as compared to cells without Nec1 or with siRNA control siLamin.

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.
3.
Figure 2

Figure 2. Coordinate autophagy and mTOR inhibition is cytotoxic to human RCC.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

(A) Viability graph of RCC4 cells treated with 10 µM or 20 µM CQ, 20 µM CCI-779 or combination of CQ and CCI-779. After 7 days, cells treated with CQ and CCI-779 as single agents were viable and showed growth arrest. The combination of 20 µM CQ with 20 µM CCI-779 reduced viability to 10%. (*p = 0.033, +p = 0.038,**p = 0.016,++p = 0.028.) (B) Viability graph of 20 µM CQ alone, 250 nM or 500 nM Torin1 or the combination of CQ and Torin1. After 7 days CQ treated cells were viable, Torin1 treated cells were 60% viable, and the combination of CQ and Torin1 were 20% viable (20 µM CQ+250 nM Torin1) and 10% viable (20 µM CQ+500 nM Torin1). Viability was measured by trypan blue exclusion and is relative to day 0. Error bars represent ± S.D. (*p = 0.020,+p = 0.036,**0.016, (C) Clonogenic survival assay showing that the combination of CQ and CCI-779 or CQ and Torin1 severely impairs long term survival. (D) % viability of knockdown cells under 40 µM CQ, 40 µM CCI-779 or combination of CQ and CCI-779 for 18 hours. CCI-779 alone with atg7 knockdown induced significant cell death (*p = 0.002 **p = 2.01×10−4). Error bars represent ± S.D. (E). RCC4 cells were grown as xenografts in nude mice. When tumors reached 150 mm3, mice were injected with vehicle (dark blue), CQ (red), CCI-779 (yellow) or combination of CQ and CCI-779 (light blue). Combination of CQ and CCI-779 prevented further tumor growth as compared to CQ alone (p = 0.011) or CCI-779 (p = 0.003). Arrows indicate days that CQ and CCI-779 were injected. Error bars are ± s.e.m.

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.
4.
Figure 1

Figure 1. The human renal cancer cell line RCC4 has high basal autophagy.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

(A) LC3 immunoblotting in human RCC cell lines. Human RCC cells have high basal autophagy as indicated by the presence of the lipidated-conjugate, LC3-II, under normal culture conditions. (B) Quantitation of LC3 punctation in RCC4 under a 9 hour drug treatment. 40 µM CQ caused accumulation of autophagosomes above basal levels (*p = 3.05×10−5) while 40 µM CCI-779 caused clearance of autophagosomes after a 9 hour drug treatment (**p = 0.0013). Error bars represent ± standard deviation (S.D.). (C) LC3 immunostaining of RCC4. Representative photos of Figure 1B. Scale bar = 10 microns. (D) Western blot of RCC4 showing accumulation of LC3-II with CQ and clearance of LC3-II with CCI-779 at 9, 12 and 16 hours. S6 lost phosphorylation with CCI-779 treatment while total S6 level remained the same. (E) Western blot showing knockdown of Atg7 in RCC4 after expression of two shRNAs targeted against Atg7 (shatg7 #1, shatg7 #2), as compared to control siRNA's, shRfps or shLUC. (F) Viability graph showing impaired viability and cell growth after atg7 knockdown. Error bars represent ± standard deviation (S.D.). (*p = 0.030 **p = 0.002).

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.
5.
Figure 5

Figure 5. mTOR inhibition blocked Nrf2 nuclear translocation and anti-oxidant defense creating dependency on autophagy for survival.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

(A) Ectopic expression of Nrf2 (red) and nuclear staining with Dapi (blue) after a 6 hour drug incubation. Yellow numbers represent the percent of cells with nuclear Nrf2 +/− the standard deviation. Scale bar = 10 µM. (B) Western blot time course of CCI-779 treated RCC4 cells. Nrf2 and p62 were induced with CCI-779 treatment, while Keap1 levels go down. S6 lost phosphorylation after 1 hour of CCI-779 treatment, while total S6 remained the same. Akt lost phosphorylation at serine 473 after 9 hours of CCI-779 treatment. Gsk3β lost its phosphorylation after 1 hour of CCI-779 incubation. (C) Immunoprecipitation of IGG control antibody, Nrf2, and Keap1 after 0, 3, 6 hours CCI-779 incubation. Nrf2 and Keap1 did not co-immunoprecipitate while p62 co-immunoprecipitates with Keap1. (D) Western blot of siRNA knockdown of Keap1. RCC4 cells were transfected with either Lamin or Keap1 siRNA and analyzed for levels of Keap1. Knockdown of Keap1 induced Nrf2. (E) Viability graph showing knockdown of Keap1 rescued cell death induced by CQ and CCI-779 combination as compared to the Lamin control after 18 hours. (*p = 0.003) (F) Clonogenic survival assay of RCC4 cells with RNA knockdown of Keap1 or Lamin control. (G) CCI-779 regulated transcriptional activity of Nrf2. RCC4 cells were transfected with the NQO1-ARE promoter firefly luciferase and Renilla luciferase as a control. Relative NQO1 mRNA level was measured. (*p = 2.353×10−5) (H) CCI-779 regulated mRNA level and protein level (I) of the Nrf2 target gene Nqo1. (Western blot of Nqo1). (*p = 0.003). (J) Localization of ectopically expressed Nrf2-RFP. CCI-779 caused exclusion Nrf2 from the nucleus. Pretreatment with a Gsk3β inhibitor restored nuclear localization of Nrf2. Yellow numbers represent % nuclear localization +/− S.D. (K) Clonogenic survival assay showing enhanced survival of CQ and CCI-779 treated cells when treated in combination with either 2 or 5 µM Gsk3β inhibitor. (L) Quantitation of colonies formed after the combination of CQ and CCI-779 with the Gsk3β inhibitor (*p = 1.01×10−4).

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.
6.
Figure 4

Figure 4. Mitochondrial ROS production causes cell death with coordinate mTOR and autophagy inhibition.. From: Autophagy Suppresses RIP Kinase-Dependent Necrosis Enabling Survival to mTOR Inhibition.

(A) Mean ROS levels in RCC4, untreated, CQ, CCI-779, or combination of CQ and CCI-779 after 6 hours by flow-cytometry using the ROS sensor DCF-DA (*p = 0.003,**p = 0.004). (B) Viability graph of RCC4 cells after 18 hours with 40 µM CQ 40 µM CCI-779, 4 nM bafilomycin A1 (BAF) or combination of CQ with CCI-779 or BAF. Cell death is prevented with the addition of 5 mM n-acetyl-cysteine (NAC) or 8 mM methyl-pyruvate (MP) (*p = 0.004,**p = 0.002,+p = 0.0003,++p = 0.0002). (C) Clonogenic survival assay of B. (D) Heat map showing relative pool sizes of TCA cycle intermediates measured by LC-MS after 3 hour drug incubation. RCC4 cells with CCI-779 and CQ plus CCI-779 showed accumulation of phosphoenolpyruvate (PEP) and a depletion of pyruvate (*p = <0.05). (E) Oxygen consumption rate (OCR) of RCC4 cells with DMEM control, 40 µM CCI-779, CCI-779+5 mM NAC or CCI-779+8 mM MP after a 3 hour drug incubation (*p = 0.002,**p = 0.012). (F) RCC4 cells were stained with the mitochondrial potential-dependent dye Mitotracker Red and Tom20 after 3 hours of drug incubation. Scale bar = 10 microns. (G) RCC4 cells expressing m-cherry-Parkin. Levels of mitophagy were quantitated after a 9 hour drug treatment by counting the percent of cells with punctuate m-cherry-Parkin expression. Scale bar = 10 microns. % colocalization of mc-Parkin and Tom 20 +/− S.D. shown as yellow numbers. (H) CCI-779 induces mitophagy (38%,*p = 9.52×10−6). Levels of mitophagy are enhanced in the combination of CQ and CCI-779 (60%,**3.752×10−5). (I) RCC4 cells pretreated for one hour with 10 µM CCCP followed by addition of CQ, CCI-779 or combination of CQ and CCI-779 to the media with and without CCCP (*p = 0.002). (J) CCCP enhanced clonogenic survival of cells treated with the combination of CQ and CCI-779 for 18 hours. (K) Western blots showing loss of RIPK1, RIPK3 and the mitochondrial marker Tom20 with CCI-779 at 12 hours, which is blocked by combination of CQ and CCI-779 as well as cleavage of the mitochondrial marker Opa1. (L) Western blot showing RIPK1 and RIPK3 degradation is prevented by 100 nm epoxomicin.

Kevin Bray, et al. PLoS One. 2012;7(7):e41831.

Display Settings:

Items per page

Supplemental Content

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...
Write to the Help Desk