Src Family Kinase Inhibitor PP2 Induces LC3 Conversion in a Manner That is Uncoupled from Autophagy and Increases Apoptosis in Multidrug-Resistant Cells

Kim, Yun-Ki; Ahn, Junho; Lee, Michael

doi:10.4062/biomolther.2012.20.4.393

Cited by 8 publications

(4 citation statements)

References 36 publications

(48 reference statements)

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“…Gossypol‐induced autophagy also seems to have a negative effect on apoptosis, since apoptosis was much more strongly induced in Ras‐NIH 3T3/Mdr cells than in Ras‐NIH 3T3 cells at 48 h. Thus, these results suggest that gossypol‐induced autophagy is cytoprotective rather than a part of the cell death process induced by gossypol. In accordance with this theory, our previous report also demonstrated that defective autophagy may contribute to the growth inhibition of Ras‐NIH 3T3/Mdr cells in response to a selective Src tyrosine kinase inhibitor, PP2 (Ahn and Lee, 2011; Kim et al, 2012). In fact, both in vitro and in vivo data fully support that autophagy can facilitate the resistance of tumor cells to anticancer treatments (Kondo et al, 2005; Chen et al, 2010).…”

Section: Discussionsupporting

confidence: 64%

“…In particular, it has been reported by Kong et al (2012) that the enhancement of autophagy in MDR cells resulted in cell survival during chemotherapy‐induced stress. In addition, we previously reported that defective autophagy might contribute to the inhibition of growth in MDR cells (Ahn and Lee, 2011; Kim et al, 2012). Indeed, clinical trials have been performed using an inhibitor of mTOR, a major negative regulator of autophagy, in combination with other anticancer agents to achieve better therapeutic outcomes (Meric‐Bernstam and Gonzalez‐Angulo, 2009).…”

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confidence: 99%

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Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

Ahn

Jang

Lee

2013

Journal Cellular Physiology

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The clinical efficacy of many chemotherapeutic agents has been reduced due to the development of drug resistance. In this article, we aimed to validate gossypol, a natural BH3 mimetic found in cottonseeds, as a potential therapeutic to overcome multidrug resistance (MDR). Gossypol was found to retain its efficacy in v-Ha-ras-transformed NIH 3T3 cells that overexpressed P-glycoprotein (Ras-NIH 3T3/Mdr), which was similar to the efficacy observed in their parental counterparts (Ras-NIH 3T3). A rhodamine assay revealed that the alteration of MDR activity did not contribute to the cytotoxic effect of gossypol. Gossypol caused a G2 /M arrest by the induction of p21(Cip1) and the down-regulation of p27(Kip1) expression in Ras-NIH 3T3 cells, whereas no significant G2 /M arrest was exhibited in Ras-NIH 3T3/Mdr cells. Surprisingly, a 48-h treatment with gossypol induced apoptotic cell death in Ras-NIH 3T3 cells; however, gossypol induced both apoptosis and necrosis in Ras-NIH 3T3/Mdr cells, as determined with flow cytometry analysis. More notably, gossypol preferentially induced autophagy in Ras-NIH 3T3 cells but not in Ras-NIH 3T3/Mdr cells. Coimmunoprecipitation and flow cytometric analysis revealed that gossypol-induced autophagy is independent of the dissociation of Beclin 1 from Bcl-2 in Ras-NIH 3T3 cells. Taken together, these results suggest that the antiproliferative activity of gossypol appears to be due to cell-cycle arrest at the G2 /M phase, with the induction of apoptosis in Ras-NIH 3T3 cells. In addition, defective autophagy might contribute to apoptotic and necrotic cell death in response to gossypol in Ras-NIH 3T3/Mdr cells.

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Section: Discussionsupporting

confidence: 64%

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confidence: 99%

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

Ahn

Jang

Lee

2013

Journal Cellular Physiology

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“…Autophagy-related cell death is characterized by the accumulation of vesicles. 23 Compound K (20 μ g/ml) triggered the accumulation of vacuolated HCT-116 cells following treatment for 24 h (Figure 2a). The vacuoles were positively stained with the lysosome marker dye, acridine orange (Figure 2b).…”

Section: Resultsmentioning

confidence: 99%

A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

et al. 2013

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Compound K (20-O-(β-D-glucopyranosyl)-20(S)-protopanaxadiol) is an active metabolite of ginsenosides and induces apoptosis in various types of cancer cells. This study investigated the role of autophagy in compound K-induced cell death of human HCT-116 colon cancer cells. Compound K activated an autophagy pathway characterized by the accumulation of vesicles, the increased positive acridine orange-stained cells, the accumulation of LC3-II, and the elevation of autophagic flux. Whereas blockade of compound K-induced autophagy by 3-methyladenein and bafilomycin A1 significantly increased cell viability. In addition, compound K augmented the time-dependent expression of the autophagy-related proteins Atg5, Atg6, and Atg7. However, knockdown of Atg5, Atg6, and Atg7 markedly inhibited the detrimental impact of compound K on LC3-II accumulation and cell vitality. Compound K-provoked autophagy was also linked to the generation of intracellular reactive oxygen species (ROS); both of these processes were mitigated by the pre-treatment of cells with the antioxidant N-acetylcysteine. Moreover, compound K activated the c-Jun NH2-terminal kinase (JNK) signaling pathway, whereas downregulation of JNK by its specific inhibitor SP600125 or by small interfering RNA against JNK attenuated autophagy-mediated cell death in response to compound K. Compound K also provoked apoptosis, as evidenced by an increased number of apoptotic bodies and sub-G1 hypodiploid cells, enhanced activation of caspase-3 and caspase-9, and modulation of Bcl-2 and Bcl-2-associated X protein expression. Notably, compound K-stimulated autophagy as well as apoptosis was induced by disrupting the interaction between Atg6 and Bcl-2. Taken together, these results indicate that the induction of autophagy and apoptosis by compound K is mediated through ROS generation and JNK activation in human colon cancer cells.

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“…It has been suggested that autophagy inhibition effectively enhances cell killing induced by the SFK inhibitor PP2 (Rothschild et al, ; Wu et al, ). We previously showed that PP2 induces LC3 conversion and inhibits growth of multidrug‐resistant NIH 3T3 cells (Kim, Ahn, & Lee, ). In the present study, we confirmed that autophagy is associated with PP2‐induced growth inhibition.…”

Section: Discussionmentioning

confidence: 99%

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

Hwang

Han

Lee

2017

Journal Cellular Physiology

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Autophagy can either promote or inhibit cell death in different cellular contexts. In this study, we investigated the role of autophagy in ATG5 knockout (KO) cell line established using CRISPR/ Cas9 system. In ATG5 KO cells, RT-PCR and immunoblot of LC3 confirmed the functional gene knockout. We found that knockout of ATG5 significantly increased proliferation of NIH 3T3 cells. In particular, autophagy deficiency enhanced susceptibility to cellular transformation as determined by an in vitro clonogenic survival assay and a soft agar colony formation assay. We also found that ATG5 KO cells had a greater migration ability as compared to wild-type (WT) cells. Moreover, ATG5 KO cells were more resistant to treatment with a Src family tyrosine kinase inhibitor (PP2) than WT cells were. Cyto-ID Green autophagy assay revealed that PP2 failed to induce autophagy in ATG5 KO cells. PP2 treatment decreased the percentage of cells in the S and G 2 /M phases among WT cells but had no effect on cell cycle distribution of ATG5 KO cells, which showed a high percentage of cells in the S and G 2 /M phases. Additionally, the proportion of apoptotic cells significantly decreased after treatment of ATG5 KO cells with PP2 in comparison with WT cells. We found that expression levels of p53 were much higher in ATG5 KO cells. The ATG5 KO seems to lead to compensatory upregulation of the p53 protein because of a decreased apoptosis rate. Taken together, our results suggest that autophagy deficiency can lead to malignant cell transformation and resistance to PP2.Autophagy is a crucial adaptive response that functions as a primary route of degradation for long-lived proteins and cytoplasmic organelles during normal cellular homeostasis (Mizushima & Komatsu, 2011). Nonetheless, there are conflicting data on the involvement of autophagy in the regulation of cell death or survival. In fact, autophagy can either promote or inhibit cell death in different cellular contexts (Maycotte & Thorburn, 2011; White et al., 2011). Our previous studies also showed that autophagy can serve either pro-survival or -death functions (Ahn, Lee, Ahn, & Lee, 2014;Kim & Lee, 2014;Kim, Han, & Lee, 2016). We recently reported a cytoprotective role of autophagy against BH3 mimetic gossypol in melanoma cells and glioma cells (Kim & Lee, 2014;Kim et al., 2016). Conversely, we found that an inhibitor (UAI-201) of oncogenic protein BRAF induces cell cycle arrest and autophagy in BRAF-mutant glioma cells (Ahn et al., 2014). In particular, deletion of ATG5 in mice results in the development of a benign tumor in the liver (Takamura et al., 2011), suggesting that autophagy has tumor-suppressive effects.Src family kinases (SFKs) are kept in an inactive state but can be switched to an active state by abnormal events such as a mutation (Okada, 2012). Active forms of SFKs are central mediators in multiple signaling pathways that are important in oncogenesis (Kim, Song, & Haura, 2009). Thus, Src inhibition has been proposed recently as a strategy for cancer therapy (Kim et al., 2...

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Src Family Kinase Inhibitor PP2 Induces LC3 Conversion in a Manner That is Uncoupled from Autophagy and Increases Apoptosis in Multidrug-Resistant Cells

Cited by 8 publications

References 36 publications

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

Defective autophagy in multidrug resistant cells may lead to growth inhibition by BH3‐mimetic gossypol

A ginseng metabolite, compound K, induces autophagy and apoptosis via generation of reactive oxygen species and activation of JNK in human colon cancer cells

Knockout of ATG5 leads to malignant cell transformation and resistance to Src family kinase inhibitor PP2

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