Acetylshikonin Induces Apoptosis in Human Colorectal Cancer HCT‐15 and LoVo Cells via Nuclear Translocation of FOXO3 and ROS Level Elevation

Lim, Heui Min; Lee, Jongsung; Nam, Myeong Jin; Park, See‐Hyoung

doi:10.1155/2021/6647107

Cited by 17 publications

(8 citation statements)

References 58 publications

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“…Previous studies represented to present that overexpression of HGF/cMet inhibited apoptosis in cancer cells via modulating mitochondrial proteins [40]. ROS-mediated apoptosis is critically associated with the intracellular ROS accumulation and regulation of the proapoptotic BH3-only proteins (Bim and Bax) and anti-apoptotic proteins (Bcl-xL) expression, which triggers mitochondrial membrane permeabilization [41,42]. Thus, we verified that transfection of Crispr-HGF and H2O2 induced apoptosis in HCC Huh7 and Hep3B cells.…”

Section: Discussionsupporting

confidence: 71%

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells

Lee

Lim

Park

et al. 2021

JPM

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Background: CRISPR/Cas9 system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Hepatocyte growth factor (HGF) is a well-known growth factor that plays a crucial role in cell growth and organ development. According to recent studies, it has been reported that HGF promoted growth of hepatocellular carcinoma (HCC) cells. Here, we investigated the apoptotic effects in HCC cells. Methods: Crispr-HGF plasmid was constructed using GeneArt CRISPR Nuclease Vector. pMex-HGF plasmid that targets HGF overexpressing gene were designed with pMex-neo plasmid. We performed real time-polymerase chain reaction to measure the expression of HGF mRNA. We performed cell counting assay and colony formation assay to evaluate cell proliferation. We also carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. Furthermore, we performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. Collectively, we performed annexin V/PI staining assay and Western blot assay. Results: In Crispr-HGF-transfected group, the mRNA expression levels of HGF were markedly downregulated compared to pMex-HGF-transfected group. Moreover, Crispr-HGF inhibited cell viability in HCC cells. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Expression of the phosphorylation of mitogen activated protein kinases and c-Met protein was regulated in Crispr-HGF-transfected group. Interestingly, we found that the expression of HGF protein in conditioned media significantly decreased in Crispr-HGF-transfected group. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh7 and Hep3B cells.

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Section: Discussionsupporting

confidence: 71%

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells

Lee

Lim

Park

et al. 2021

JPM

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“…Primary antibodies were diluted to 1 : 500~1000 ratio, and secondary antibodies were diluted to 1 : 5000 ratio for use. All the experimental protocols were referred from our previous publications [ 13 , 45 , 46 ].…”

Section: Methodsmentioning

confidence: 99%

“…Therefore, acetylshikonin was assessed as an alternative and the result was pleasable that acetylshikonin had much less toxicity than shikonin in normal cells with corresponding antigenotoxic effect [ 12 ]. Furthermore, our previous study demonstrated that cytotoxicity was negligible in normal cells up to 5 μ M of concentration [ 13 ]. The anticancer effects of acetylshikonin is emerging in various cancer cells [ 14 ] and identified as the novel CYP2J2 inhibitor in hepatocellular carcinoma HepG2 cells [ 15 ], but the effects in RCC were not been investigated yet.…”

Section: Introductionmentioning

confidence: 99%

Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation

Lim

Lee

et al. 2022

Oxidative Medicine and Cellular Longevity

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Acetylshikonin is a shikonin derivative originated from Lithospermum erythrorhizon roots that exhibits various biological activities, including granulation tissue formation, promotion of inflammatory effects, and inhibition of angiogenesis. The anticancer effect of acetylshikonin was also investigated in several cancer cells; however, the effect against renal cell carcinoma (RCC) have not yet been studied. In this study, we aimed to investigate the anticarcinogenic mechanism of acetylshikonin in A498 and ACHN, human RCC cell lines. MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide), cell counting, and colony forming assay showed that acetylshikonin induced cytotoxic and antiproliferative effects in a dose- and time-dependent manner. Cell cycle analysis and annexin V/propidium iodide (PI) double staining assay indicated the increase of subG1 phase and apoptotic rates. Also, DNA fragmentation was observed by using the TUNEL and comet assays. The intracellular ROS level in acetylshikonin-treated RCC was evaluated using DCF-DA. The ROS level was increased and cell viability was decreased in a dose- and time-dependent manner, while those were recovered when cotreated with NAC. Western blotting analysis showed that acetylshikonin treatment increased the expression of FOXO3, cleaved PARP, cleaved caspase-3, -6, -7, -8, -9, γH2AX, Bim, Bax, p21, and p27 while decreased the expressions of CYP2J2, peroxiredoxin, and thioredoxin-1, Bcl-2, and Bcl-xL. Simultaneously, nuclear translocation of FOXO3 and p27 was observed in cytoplasmic and nuclear fractionated western blot analysis. Acetylshikonin was formerly identified as a novel inhibitor of CYP2J2 protein in our previous study and it was evaluated that CYP2J2 was downregulated in acetylshikonin-treated RCC. CYP2J2 siRNA transfection augmented that apoptotic effect of acetylshikonin in A498 and ACHN via up-regulation of FOXO3 expression. In conclusion, we showed that the apoptotic potential of acetylshikonin against RCC is mediated via increase of intracellular ROS level, activation of FOXO3, and inhibition of CYP2J2 expressions. This study offers that acetylshikonin may be a considerable alternative therapeutic option for RCC treatment by targeting FOXO3 and CYP2J2.

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“…ASH induces apoptosis in a wide range of tumor cells, such as ROS-dependent apoptosis in oral squamous cell carcinoma cells (Ca9-22) [7]. Recent investigations have shown that ASH increases ROS and nuclear damage with subsequent nuclear translocation of FOXO3 and induced caspase-dependent apoptosis in osteosarcoma U2OS, renal cell carcinoma and colorectal cancer HCT-15, and LoVo cells [85][86][87]. It has also been reported that ASH induces dose-dependent apoptosis via activation of caspase-3/-7 and -9 in chondrosarcoma cell lines [88].…”

Section: Discussionmentioning

confidence: 99%

“…Previous investigations have shown that an increase of pro-apoptotic Bcl2 family proteins or decrease in anti-apoptotic Bcl2 protein expression might be involved in mitochondrial damage, increase in cellular ROS and decrease in mitochondrial membrane potential [31,46,49,70,[91][92][93]. It has been showed that ASH induces apoptosis in osteosarcoma U2OS cells , A498 and ACHN (human RCC cell lines), colorectal Cancer HCT-15 and LoVo cells, and human leukemia cell line K562, via changing the Bcl2 family proteins [85][86][87]89]. Interestingly, our investigations showed that ASH did not significantly change Bax, Bcl2, Bcl-XL and Mcl-1 expression while it decreased mitochondrial membrane potential, increased ROS, activated caspase-3/-7 and induced apoptosis in GBM cell lines (U87 and U251).…”

Section: Discussionmentioning

confidence: 99%

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial Dependent Apoptosis in GBM Cells which is Regulated by Autophagy

Hajiahmadi¹,

Lorzadeh²,

Iranpour³

et al. 2023

Preprint

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Glioblastoma multiforme (GBM) is one of the deadliest cancers. Temozolomide (TMZ) is the most common chemotherapy used for GBM patients. Recently, combination chemotherapy strategies have more effective antitumor effects and focus on slowing down the development of chemotherapy resistance. A combination of TMZ and cholesterol lowering medications (statins) is currently under investigation in in vivo and clinical trials. In our current investigation, we have used a triple combination therapy of TMZ, Simvastatin (Simva), and Acetylshikonin (ASH) and investigated its apoptotic mechanism in GBM cell lines (U87 and U251). We used viability, apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), caspase-3/-7, acridine orange (AO) and immunoblotting autophagy assays. Our results showed that TMZ/Simva/ASH combination therapy significantly induced more apoptosis compared to TMZ, Simva, ASH, and TMZ/Simva treatments in GBM cells. Apoptosis via TMZ/Simva/ASH treatment induced mitochondrial damage (increase of ROS, decrease of MMP) and induced caspase-3/7 activation in both GBM cell lines. Compared to all single treatments and the TMZ/Simva treatment, TMZ/Simva/ASH significantly increased positive acidic vacuole organelles. We further confirmed that the increase of AVOs during the TMZ/Simva/ASH treatment was due to partial inhibition of autophagy flux (accumulation of LC3β-II and decrease in p62 degradation) in GBM cells. Our investigation also showed that TMZ/Simva/ASH-induced cell death was depended on autophagy flux as further inhibition of autophagy flux increased TMZ/Simva/ASH-induced cell death in GBM cells. Finally, our results showed that TMZ/Simva/ASH treatment potentially depends on an increase of Bax expression in GBM cells. Our current investigation might open new avenues for more effective treatment of GBM but further investigations are required for better identification of the mechanisms.

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Acetylshikonin Induces Apoptosis in Human Colorectal Cancer HCT‐15 and LoVo Cells via Nuclear Translocation of FOXO3 and ROS Level Elevation

Cited by 17 publications

References 58 publications

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells

Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells

Acetylshikonin, A Novel CYP2J2 Inhibitor, Induces Apoptosis in RCC Cells via FOXO3 Activation and ROS Elevation

Temozolomide, Simvastatin and Acetylshikonin Combination Induces Mitochondrial Dependent Apoptosis in GBM Cells which is Regulated by Autophagy

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