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Autophagy inhibition enhances isorhamnetin‑induced mitochondria‑dependent apoptosis in non‑small cell lung cancer cells.

Ruan Y, Hu K, Chen H - Mol Med Rep (2015)

Bottom Line: ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner.Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo.In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Disease, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.

ABSTRACT
Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

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Autophagy inhibition enhances the growth inhibitory effect of ISO on A549 xenograft tumors. (A) Images of harvested tumors at the end of the experiment. (B) Weights of tumors from the mice after two weeks of indicated treatments. (C) Representative immunohistochemical staining for PCNA and c-caspase-3 as well as TUNEL staining (scale bar, 50 μm). (D) The proliferative and apoptotic index was calculated by the ratio of positive cells vs. total cells. Values are expressed as the mean ± standard deviation (n=6). **P<0.01. PCNA, Proliferating cell nuclear antigen; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; CQ, hydroxychloroquine; ISO, isorhamnetin; MA, methyladenine; C-caspase, cleaved caspase.
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f6-mmr-12-04-5796: Autophagy inhibition enhances the growth inhibitory effect of ISO on A549 xenograft tumors. (A) Images of harvested tumors at the end of the experiment. (B) Weights of tumors from the mice after two weeks of indicated treatments. (C) Representative immunohistochemical staining for PCNA and c-caspase-3 as well as TUNEL staining (scale bar, 50 μm). (D) The proliferative and apoptotic index was calculated by the ratio of positive cells vs. total cells. Values are expressed as the mean ± standard deviation (n=6). **P<0.01. PCNA, Proliferating cell nuclear antigen; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; CQ, hydroxychloroquine; ISO, isorhamnetin; MA, methyladenine; C-caspase, cleaved caspase.

Mentions: Confirmed by the marked anti-proliferative and apoptosis-inducing activities observed in cell culture experiments, the present study examined the anti-tumor activity of ISO in vivo. BALB/c nu/nu mice bearing A549 NSCLC xenografts were given a daily intraperitoneal injection of ISO. In a preliminary study, ISO showed an in vivo anti-tumor activity at 0.5 mg/kg/day, and this dose was therefore used in the present study. The growth of xenografts was monitored every three days over two weeks. Side effects, including body weight loss, mortality and lethargy were not observed in mice treated by ISO for two weeks. The final tumor size was markedly lower in the majority of the 0.5 mg/kg ISO-treated mice compared with that in the control group. Of note, the tumor size was significantly lower in the group co-injected with 3-MA (22.4 mg/kg) or CQ (10 mg/kg) (Fig. 6A), compared with that in the mice injected with ISO only. The tumor weight was 2.11±0.35 g in the control mice, 0.91±0.27 g in ISO-treated mice, 0.42±0.12 g in ISO and 3-MA co-injected mice and 0.58±0.16 in ISO and CQ co-injected mice, respectively (Fig. 6B). The results therefore indicated that autophagy inhibition markedly promoted the inhibitory effect of ISO on the NSCLC xenograft tumors.


Autophagy inhibition enhances isorhamnetin‑induced mitochondria‑dependent apoptosis in non‑small cell lung cancer cells.

Ruan Y, Hu K, Chen H - Mol Med Rep (2015)

Autophagy inhibition enhances the growth inhibitory effect of ISO on A549 xenograft tumors. (A) Images of harvested tumors at the end of the experiment. (B) Weights of tumors from the mice after two weeks of indicated treatments. (C) Representative immunohistochemical staining for PCNA and c-caspase-3 as well as TUNEL staining (scale bar, 50 μm). (D) The proliferative and apoptotic index was calculated by the ratio of positive cells vs. total cells. Values are expressed as the mean ± standard deviation (n=6). **P<0.01. PCNA, Proliferating cell nuclear antigen; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; CQ, hydroxychloroquine; ISO, isorhamnetin; MA, methyladenine; C-caspase, cleaved caspase.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581743&req=5

f6-mmr-12-04-5796: Autophagy inhibition enhances the growth inhibitory effect of ISO on A549 xenograft tumors. (A) Images of harvested tumors at the end of the experiment. (B) Weights of tumors from the mice after two weeks of indicated treatments. (C) Representative immunohistochemical staining for PCNA and c-caspase-3 as well as TUNEL staining (scale bar, 50 μm). (D) The proliferative and apoptotic index was calculated by the ratio of positive cells vs. total cells. Values are expressed as the mean ± standard deviation (n=6). **P<0.01. PCNA, Proliferating cell nuclear antigen; TUNEL, Terminal deoxynucleotidyl transferase dUTP nick end labeling; CQ, hydroxychloroquine; ISO, isorhamnetin; MA, methyladenine; C-caspase, cleaved caspase.
Mentions: Confirmed by the marked anti-proliferative and apoptosis-inducing activities observed in cell culture experiments, the present study examined the anti-tumor activity of ISO in vivo. BALB/c nu/nu mice bearing A549 NSCLC xenografts were given a daily intraperitoneal injection of ISO. In a preliminary study, ISO showed an in vivo anti-tumor activity at 0.5 mg/kg/day, and this dose was therefore used in the present study. The growth of xenografts was monitored every three days over two weeks. Side effects, including body weight loss, mortality and lethargy were not observed in mice treated by ISO for two weeks. The final tumor size was markedly lower in the majority of the 0.5 mg/kg ISO-treated mice compared with that in the control group. Of note, the tumor size was significantly lower in the group co-injected with 3-MA (22.4 mg/kg) or CQ (10 mg/kg) (Fig. 6A), compared with that in the mice injected with ISO only. The tumor weight was 2.11±0.35 g in the control mice, 0.91±0.27 g in ISO-treated mice, 0.42±0.12 g in ISO and 3-MA co-injected mice and 0.58±0.16 in ISO and CQ co-injected mice, respectively (Fig. 6B). The results therefore indicated that autophagy inhibition markedly promoted the inhibitory effect of ISO on the NSCLC xenograft tumors.

Bottom Line: ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner.Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo.In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Division of Respiratory Disease, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China.

ABSTRACT
Isorhamnetin (ISO) is a flavonoid from plants of the Polygonaceae family and is also an immediate metabolite of quercetin in mammals. To date, the anti‑tumor effects of ISO and the underlying mechanisms have not been elucidated in lung cancer cells. The present study investigated the inhibitory effects of ISO on the growth of human lung cancer A549 cells. Treatment of the lung cancer cells with ISO significantly suppressed cell proliferation and colony formation. ISO treatment also resulted in a significant increase in apoptotic cell death of A549 cells in a time- and dose-dependent manner. Further investigation showed that the apoptosis proceeded via the mitochondria‑dependent pathway as indicated by alteration of the mitochondrial membrane potential, the release of cytochrome C and caspase activation. Of note, treatment with ISO also induced the formation of autophagosomes and light chain 3‑II protein in A549 cells. Furthermore, co‑treatment with autophagy inhibitors 3‑methyladenine and hydroxychloroquine significantly inhibited the ISO‑induced autophagy and enhanced the ISO‑induced apoptotic cell death in vitro as well as in vivo. Thus, the results of the present study suggested that ISO is a potential anti‑lung cancer agent. In addition, the results indicated that the inhibition of autophagy may be a useful strategy for enhancing the chemotherapeutic effect of ISO on lung cancer cells.

Show MeSH
Related in: MedlinePlus