Enhancement of auranofin-induced lung cancer cell apoptosis by selenocystine,a natural inhibitor of TrxR1 in vitro and in vivo

Thioredoxin reductase (TrxR) is overpressed in many human tumors and has a key role in regulating intracellular redox balance. Recently, thioredoxin system has emerged as a valuable target for anticancer drug development. Herein we demonstrate that selenocystine (SeC) could enhance auranofin (AF)-induced A549 human lung adenocarcinoma cell apoptosis in vitro and in vivo through synergetic inhibition of TrxR1. SeC pretreatment significantly enhanced AF-induced loss of mitochondrial membrane potential (Δψ_m) by regulating Bcl-2 family proteins. The combined treatment with SeC and AF also resulted in enhanced intracellular reactive oxygen species (ROS) accumulation, DNA damage, and inactivation of ERK and AKT. Inhibitors of ERK and AKT effectively enhanced combined treatment-induced apoptotic cell death. However, inhibition of ROS reversed the apoptosis induced by SeC and AF, and recovered the inactivation of ERK and AKT, which revealed the importance of ROS in cell apoptosis and regulation of ERK and AKT pathways. Moreover, xenograft lung tumor growth in nude mice was more effectively inhibited by combined treatment with SeC and AF by induction of apoptosis through targeting TrxR1 in vivo. Taken together, our results suggest the strategy to use SeC and AF in combination could be a highly efficient way to achieve anticancer synergism by targeting TrxR1.     

Potent antitumor activities of recombinant human PDCD5 protein in combination with chemotherapy drugs in K562 cells

Conventional chemotherapy is still frequently used. Programmed cell death 5 (PDCD5) enhances apoptosis of various tumor cells triggered by certain stimuli and is lowly expressed in leukemic cells from chronic myelogenous leukemia patients. Here, we describe for the first time that recombinant human PDCD5 protein (rhPDCD5) in combination with chemotherapy drugs has potent antitumor effects on chronic myelogenous leukemia K562 cells in vitro and in vivo. The antitumor efficacy of rhPDCD5 protein with chemotherapy drugs, idarubicin (IDR) or cytarabine (Ara-C), was examined in K562 cells in vitro and K562 xenograft tumor models in vivo. rhPDCD5 protein markedly increased the apoptosis rates and decreased the colony-forming capability of K562 cells after the combined treatment with IDR or Ara-C. rhPDCD5 protein by intraperitoneal administration dramatically improved the antitumor effects of IDR treatment in the K562 xenograft model. The tumor sizes and cell proliferation were significantly decreased; and TUNEL positive cells were significantly increased in the combined group with rhPDCD5 protein and IDR treatment compared with single IDR treatment groups. rhPDCD5 protein, in combination with IDR, has potent antitumor effects on chronic myelogenous leukemia K562 cells and may be a novel and promising agent for the treatment of chronic myelogenous leukemia.     

A novel Osmium-based compound targets the mitochondria and triggers ROS-dependent apoptosis in colon carcinoma

Engagement of the mitochondrial-death amplification pathway is an essential component in chemotherapeutic execution of cancer cells. Therefore, identification of mitochondria-targeting agents has become an attractive avenue for novel drug discovery. Here, we report the anticancer activity of a novel Osmium-based organometallic compound (hereafter named Os) on different colorectal carcinoma cell lines. HCT116 cell line was highly sensitive to Os and displayed characteristic features of autophagy and apoptosis; however, inhibition of autophagy did not rescue cell death unlike the pan-caspase inhibitor z-VAD-fmk. Furthermore, Os significantly altered mitochondrial morphology, disrupted electron transport flux, decreased mitochondrial transmembrane potential and ATP levels, and triggered a significant increase in reactive oxygen species (ROS) production. Interestingly, the sensitivity of cell lines to Os was linked to its ability to induce mitochondrial ROS production (HCT116 and RKO) as HT29 and SW620 cell lines that failed to show an increase in ROS were resistant to the death-inducing activity of Os. Finally, intra-peritoneal injections of Os significantly inhibited tumor formation in a murine model of HCT116 carcinogenesis, and pretreatment with Os significantly enhanced tumor cell sensitivity to cisplatin and doxorubicin. These data highlight the mitochondria-targeting activity of this novel compound with potent anticancer effect in vitro and in vivo, which could have potential implications for strategic therapeutic drug design.     

Molecular evidence of anti-leukemia activity of gypenosides on human myeloid leukemia HL-60 cells in vitro and in vivo using a HL-60 cells murine xenograft model

We have shown that gypenosides (Gyp) induced cell cycle arrest and apoptosis in many human cancer cell lines. However, there are no reports showing that show Gyp acts on human leukemia HL-60 cells in vitro and in a murine xenograft model in vivo. In the present study effects of Gyp on cell morphological changes and viability, cell cycle arrest and induction of apoptosis in vitro and effects on Gyp in an in vivo murine xenograft model. Results indicated that Gyp induced morphological changes, decreased cell viability, induced G0/G1 arrest, DNA fragmentation and apoptosis (sub-G1 phase) in HL-60 cells. Gyp increased reactive oxygen species production and Ca²⁺ levels but reduced mitochondrial membrane potential in a dose- and time-dependent manner. Gyp also changed one of the primary indicators of endoplasmic reticulum (ER) stress due to the promotion of ATF6-α and ATF4-α associated with Ca²⁺ release. Gyp reduced the ratio of Bcl-2 to Bax due to an increase in the pro-apoptotic protein Bax and inhibited levels of the anti-apoptotic protein Bcl-2. Oral consumption of Gyp reduced tumor size of HL-60 cell xenograft mode mice in vivo. These results provide new information on understanding mechanisms by which Gyp induces cell cycle arrest and apoptosis in vitro and in vivo.     

Shikonin inhibits proliferation of melanoma cells by MAPK pathway-mediated induction of apoptosis

Shikonin, a natural product isolated from the roots of Lithospermum erythrorhizon, exhibits pharmacological effects against inflammation, ulcers, infections, and tumors. In the present study, we aimed to investigate the antitumor effects of shikonin on the human melanoma cell line, A375SM, and in an in vivo mouse xenograft model. We examined the anticancer effects of shikonin by in vitro experiments (MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, 4′,6-diamidino-2-phenylindole (DAPI) stain, annexin V/ propidium iodide (PI) stain, and protein analysis of apoptosis and mitogen-activated protein kinase (MAPK) pathways). Further, the anticancer effect in vivo was confirmed through a xenograft model. Our results showed that shikonin inhibited the proliferation of melanoma cells in a dose-dependent manner. In addition, shikonin significantly increased nucleus and chromatin condensation and early/late apoptosis. Shikonin also increased the pro-apoptotic proteins and decreased the anti-apoptotic proteins. Additionally, shikonin was overexpressed in MAPK pathways. Investigation of the effects of shikonin in a mouse xenograft model not only showed decreased A375SM tumor volume but also increased apoptosis as determined by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Furthermore, pathologic changes were not observed in the liver and kidney of mice. Collectively, the study indicated that shikonin inhibited the proliferation of the human melanoma cells by inducing apoptosis, mediated by MAPK pathway and that it is a potential candidate for an anticancer drug against melanoma cancer.     

Functional Characterisation of Anticancer Activity in the Aqueous Extract of Helicteres angustifolia L. Roots

Helicteres angustifolia L. is a shrub that forms a common ingredient of several cancer treatment recipes in traditional medicine system both in China and Laos. In order to investigate molecular mechanisms of its anticancer activity, we prepared aqueous extract of Helicteres angustifolia L. Roots (AQHAR) and performed several in vitro assays using human normal fibroblasts (TIG-3) and osteosarcoma (U2OS). We found that AQHAR caused growth arrest/apoptosis of U2OS cells in a dose-dependent manner. It showed no cytotoxicity to TIG-3 cells at doses up to 50 μg/ml. Biochemical, imaging and cell cycle analyses revealed that it induces ROS signaling and DNA damage response selectively in cancer cells. The latter showed upregulation of p53, p21 and downregulation of Cyclin B1 and phospho-Rb. Furthermore, AQHAR-induced apoptosis was mediated by increase in pro-apoptotic proteins including cleaved PARP, caspases and Bax. Anti-apoptotic protein Bcl-2 showed decrease in AQHAR-treated U2OS cells. In vivo xenograft tumor assays in nude mice revealed dose-dependent suppression of tumor growth and lung metastasis with no toxicity to the animals suggesting that AQHAR could be a potent and safe natural drug for cancer treatment.     

Shuganning injection,a traditional Chinese patent medicine,induces ferroptosis and suppresses tumor growth in triple-negative breast cancer cells

BackgroundTriple-negative breast cancer (TNBC), lacking targeted therapies currently, is susceptible to ferroptosis, a recently defined form of cell death.PurposeTo evaluate the anticancer activity of Shuganning injection (SGNI), a traditional Chinese patent medicine, on TNBC cells; To elucidate the mechanism of SGNI induced ferroptosis.MethodsThe anticancer activity of SGNI was examined via in vitro cell proliferation assays and in vivo xenograft growth assay. Ferroptosis was determined by flow-cytometric analysis of lipid ROS, labile iron pool measurement, and propidium iodide exclusion assay. The dependency on heme oxygenase 1 (HO-1) of SGNI induced ferroptosis was confirmed by genetic knockdown and pharmacological inhibition of the protein.ResultsSGNI selectively inhibited the proliferation of TNBC cells compared to non-TNBC breast cancer cells and normal cells. The cell death induced by SGNI in TNBC cells showed distinct morphology from apoptosis and could not be rescued by the pan-caspase inhibitor Z-VAD(OMe)-FMK. On the other hand, SGNI induced cell death was blocked by the lipid ROS scavengers ferrostatin-1 and liproxstatin-1, the acyl-CoA synthetase long chain family member 4 inhibitor rosiglitazone, and the iron chelators 1,10-phenanthroline and deferoxamine. These data indicated that SGNI induced a ferroptotic cell death of TNBC cells. Mechanistically, SGNI induced ferroptosis was dependent on HO-1, which promotes intracellular labile iron pool accumulation, and was alleviated by HO-1 knockdown and inhibition by tin protoporphyrin IX. In line with the in vitro data, SGNI significantly inhibited the xenograft growth of TNBC cell line MD-MB-231 in nude mice.ConclusionCollectively, our study elaborates on a promising regimen for TNBC treatment through induction of ferroptosis by SGNI, a traditional Chinese patent medicine currently available in the clinic, which merits further investigation.     

Cotreatment with apicidin overcomes TRAIL resistance via inhibition of Bcr-Abl signaling pathway in K562 leukemia cells

TNF-related apoptosis-inducing ligand (TRAIL) is a pro-apoptotic cytokine that is capable of inducing apoptosis in a wide variety of cancer cells but not in normal cells. Although many cancer cells are sensitive to TRAIL-induced apoptosis, chronic myeloid leukemia (CML) develops resistance to TRAIL. In this study, we investigated whether apicidin, a novel histone deacetylase inhibitor, could overcome the TRAIL resistance in CML-derived K562 cells. Compared to treatment with apicidin or TRAIL alone, cotreatment with apicidin and TRAIL-induced apoptosis synergistically in K562 cells. This combination led to activation of caspase-8 and Bcl-2 interacting domain (Bid), resulting in the cytosolic accumulation of cytochrome c from mitochondria as well as an activation of caspase-3. Treatment with apicidin resulted in down-regulation of Bcr-Abl and inhibition of its downstream target, PI3K/AKT-NF-κB pathway. In addition, apicidin decreased the level of NF-κB-dependent Bcl-x_L, leading to caspase activation and Bid cleavage. These results suggest that apicidin may sensitize K562 cells to TRAIL-induced apoptosis through caspase-dependent mitochondrial pathway by regulating expression of Bcr-Abl and its related anti-apoptotic proteins. Therefore, the present study suggests that combination of apicidin and TRAIL may be an effective strategy for treating TRAIL-resistant Bcr-Abl expressing CML cells.     

XJW20, a novel oxoindole derivative,induces G2/M arrest and apoptosis selectively in K562 leukemia cell line

In comparison with four tumor cell lines and three non transformed cell types, chronic myeloid leukemia K562 cells were selectively sensitive to proliferation inhibition by the oxoindole derivative XJW20, as determined by the MTT assay. Further investigation revealed that XJW20 selectively induced G2/M arrest and apoptosis in K562 cells. At the molecular level, XJW20-induced G2/M arrest was accompanied by up-regulation of cyclin B1 and phospho (p)-Cdc25C (Ser216) and down-regulation of CDK1. There is no change in the expression of CDK2. The increased apoptotic activity by XJW20 was characterized by an increase in reactive oxygen species (ROS) generation, the mitochondrial transmembrane potential (ΔΨ_m) dissipation, cytochrome C releasing, apoptotic nuclei (AO/EB double staining) and nuclei condensation (DAPI-staining). The down-regulation of phosphorylated ERK was also found in XJW20-treated K562 cells. These molecular events induced by XJW20 may provide insight into the mechanism of action that led to growth arrest and apoptosis.     

Cartilage polysaccharide induces apoptosis in human leukemia K562 cells

In this study, we extracted a polysaccharide (short-chain polysaccharide [PS]) from porcine cartilage and examined its function in chronic myeloid leukaemia by using human K562 cells and mouse L1210 cells. Results of cell proliferation assay indicated that PS inhibited cancer cell growth at different concentrations, while it had little effect on normal cells. The presence of morphological aspects of apoptosis, such as nuclear shrinkage, was shown in H&E stained sections. The occurrence of PS-induced apoptosis was confirmed by TUNEL assay and cell cycle analysis. The results of immunofluorescent staining indicated the molecular mechanism underlying. Through interfering with the cell cycle of tumor cells, PS may induce apoptosis by downregulating the expression level of cyclin D1 and upregulating the level of p21 protein. Correlation analysis of apoptosis and MAPK suggested that inactivation of ERK was crucial for PS induced apoptosis, while JNK phosphorylation had a small effect and p38 was not involved. In vivo assay showed that PS inhibited L1210 cell growth in vivo and prolonged the life span of L1210-bearing mice. We conclude that PS is a polysaccharide with anticancer effects and induced apoptosis in human K562 cells.     

A diterpenoid derivate compound targets selenocysteine of thioredoxin reductases and induces Bax/Bak-independent apoptosis

We have previously shown that the natural diterpenoid derivative S3 induced Bim upregulation and apoptosis in a Bax/Bak-independent manner. However, the exact molecular target(s) of S3 and the mechanism controlling Bim upregulation are still not clear. Here, we identify that S3 targets the selenoproteins TrxR1 and TrxR2 at the selenocysteine residue of the reactive center of the enzymes and inhibits their antioxidant activities. Consequently, cellular ROS is elevated, leading to the activation of FOXO3a, which contributes to Bim upregulation in Bax/Bak-deficient cells. Moreover, S3 retards tumor growth in subcutaneous xenograft tumors by inhibiting TrxR activity in vivo. Our studies delineate the signaling pathway controlling Bim upregulation, which results in Bax/Bak-independent apoptosis and provide evidence that the compounds can act as anticancer agents based on mammalian TrxRs inhibition.     

Anti-Tumor Activity of Eurycoma longifolia Root Extracts against K-562 Cell Line: In Vitro and In Vivo Study

Eurycoma longifolia Jack has been widely used in traditional medicine for its antimalarial, aphrodisiac, anti-diabetic, antimicrobial and anti-pyretic activities. Its anticancer activity has also been recently reported on different solid tumors, however no anti-leukemic activity of this plant has been reported. Thus the present study assesses the in vitro and in vivo anti-proliferative and apoptotic potentials of E. longifolia on K-562 leukemic cell line. The K-562 cells (purchased from ATCC) were isolated from patients with chronic myelocytic leukemia (CML) were treated with the various fractions (TAF273, F3 and F4) of E. longifolia root methanolic extract at various concentrations and time intervals and the anti-proliferative activity assessed by MTS assay. Flow cytometry was used to assess the apoptosis and cell cycle arrest. Nude mice injected subcutaneously with 10⁷ K-562 cells were used to study the anti-leukemic activity of TAF273 in vivo. TAF273, F3 and F4 showed various degrees of growth inhibition with IC50 values of 19, 55 and 62 µg/ml, respectively. TAF273 induced apoptosis in a dose and time dependent manner. TAF273 arrested cell cycle at G1and S phases. Intraperitoneal administration of TAF273 (50 mg/kg) resulted in a significant growth inhibition of subcutaneous tumor in TAF273-treated mice compared with the control mice (P = 0.024). TAF273 shows potent anti-proliferative activity in vitro and in vivo models of CML and therefore, justifies further efforts to define more clearly the potential benefits of using TAF273 as a novel therapeutic strategy for CML management.     

Antitumor Efficacy of the Dual PI3K/mTOR Inhibitor PF-04691502 in a Human Xenograft Tumor Model Derived from Colorectal Cancer Stem Cells Harboring a PIK3CA Mutation

PIK3CA (phosphoinositide-3-kinase, catalytic, alpha polypeptide) mutations can help predict the antitumor activity of phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) pathway inhibitors in both preclinical and clinical settings. In light of the recent discovery of tumor-initiating cancer stem cells (CSCs) in various tumor types, we developed an in vitro CSC model from xenograft tumors established in mice from a colorectal cancer patient tumor in which the CD133+/EpCAM+ population represented tumor-initiating cells. CD133+/EpCAM+ CSCs were enriched under stem cell culture conditions and formed 3-dimensional tumor spheroids. Tumor spheroid cells exhibited CSC properties, including the capability for differentiation and self-renewal, higher tumorigenic potential and chemo-resistance. Genetic analysis using an OncoCarta™ panel revealed a PIK3CA (H1047R) mutation in these cells. Using a dual PI3K/mTOR inhibitor, PF-04691502, we then showed that blockage of the PI3K/mTOR pathway inhibited the in vitro proliferation of CSCs and in vivo xenograft tumor growth with manageable toxicity. Tumor growth inhibition in mice was accompanied by a significant reduction of phosphorylated Akt (pAKT) (S473), a well-established surrogate biomarker of PI3K/mTOR signaling pathway inhibition. Collectively, our data suggest that PF-04691502 exhibits potent anticancer activity in colorectal cancer by targeting both PIK3CA (H1047R) mutant CSCs and their derivatives. These results may assist in the clinical development of PF-04691502 for the treatment of a subpopulation of colorectal cancer patients with poor outcomes.     

Activation of c-Met and Upregulation of CD44 Expression Are Associated with the Metastatic Phenotype in the Colorectal Cancer Liver Metastasis Model

Background

Liver metastasis is the most common cause of death in patients with colorectal cancer. Despite extensive research into the biology of cancer progression, the molecular mechanisms that drive colorectal cancer metastasis are not well characterized.

Methods

HT29 LM1, HT29 LM2, HT29 LM3 cell lines were derived from the human colorectal cancer cell line HT29 following multiple rounds of in vivo selection in immunodeficient mice.

Results

CD44 expression, a transmembrane glycoprotein involved in cell-cell and cell-matrix adhesions, and cancer cells adhesion to endothelial cells was increased in all in vivo selected cell lines, with maximum CD44 expression and cancer cells adhesion to endothelial cells in the highly metastatic HT29 LM3 cell line. Activation of c-Met upon hepatocyte growth factor (HGF) stimulation in the in vivo selected cell lines is CD44 independent. In vitro separation of CD44 high and low expression cells from HT29 LM3 cell line with FACS sorting confirmed that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation. Furthermore, in vivo evaluation of CD44 low and high expressing HT29 LM3 cells demonstrated no difference in liver metastasis penetrance.

Conclusions

Taken together, our findings indicate that the aggressive metastatic phenotype of in vivo selected cell lines is associated with overexpression of CD44 and activation of c-MET. We demonstrate that c-Met activation is CD44 independent upon hepatocyte growth factor stimulation and confirm that CD44 expression in HT29 LM3 cell line is not responsible for the increase in metastatic penetrance in HT29 LM3 cell line.     

A Novobiocin Derivative,XN4, Inhibits the Proliferation of Chronic Myeloid Leukemia Cells by Inducing Oxidative DNA Damage

XN4 might induce DNA damage and apoptotic cell death through reactive oxygen species (ROS). The inhibition of proliferation of K562 and K562/G01 cells was measured by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide). The mRNA levels of NADPH oxidase 1-5 (Nox1-5) genes were evaluated by qRT-PCR. The levels of extracellular reactive oxygen species (ROS), DNA damage, apoptosis, and cell cycle progression were examined by flow cytometry (FCM). Protein levels were analyzed by immunoblotting. XN4 significantly inhibited the proliferation of K562 and K562/G01 cells, with IC₅₀ values of 3.75±0.07 µM and 2.63±0.43 µM, respectively. XN4 significantly increased the levels of Nox4 and Nox5 mRNA, stimulating the generation of intracellular ROS, inducing DNA damage and activating ATM-γ-H2AX signaling, which increased the number of cells in the S and G2/M phase of the cell cycle. Subsequently, XN4 induced apoptotic cell death by activating caspase-3 and PARP. Moreover, the above effects were all reversed by the ROS scavenger N-acetylcysteine (NAC). Additionally, XN4 can induce apoptosis in progenitor/stem cells isolated from CML patients’ bone marrow. In conclusion, XN4-induced DNA damage and cell apoptosis in CML cells is mediated by the generation of ROS.     

Dramatic suppression of colorectal cancer cell growth by the dual mTORC1 and mTORC2 inhibitor AZD-2014

Colorectal cancer is a major contributor of cancer-related mortality. The mammalian target or rapamycin (mTOR) signaling is frequently hyper-activated in colorectal cancers, promoting cancer progression and chemo-resistance. In the current study, we investigated the anti-colorectal cancer effect of a novel mTOR complex 1 (mTORC1) and mTORC2 dual inhibitor: AZD-2014. In cultured colorectal cancer cell lines, AZD-2014 significantly inhibited cancer cell growth without inducing significant cell apoptosis. AZD-2014 blocked activation of both mTORC1 (S6K and S6 phosphorylation) and mTORC2 (Akt Ser 473 phosphorylation), and activated autophagy in colorectal cancer cells. Meanwhile, autophagy inhibition by 3-methyaldenine (3-MA) and hydroxychloroquine, as well as by siRNA knocking down of Beclin-1 or ATG-7, inhibited AZD-2014-induced cytotoxicity, while the apoptosis inhibitor had no rescue effect. In vivo, AZD-2014 oral administration significantly inhibited the growth of HT-29 cell xenograft in SCID mice, and the mice survival was dramatically improved. At the same time, in xenografted tumors administrated with AZD-2014, the activation of mTORC1 and mTORC2 were largely inhibited, and autophagic markers were significantly increased. Thus, AZD-2014 inhibits colorectal cancer cell growth both in vivo and in vitro. Our results suggest that AZD-2014 may be further investigated for colorectal cancer therapy in clinical trials.     

PAX9 reactivation by inhibiting DNA methyltransferase triggers antitumor effect in oral squamous cell carcinoma

Aberrant DNA hypermethylation is associated with oral carcinogenesis. Procaine, a local anesthetic, is a DNA methyltransferase (DNMT) inhibitor that activates anticancer mechanisms. However, its effect on silenced tumor suppressor gene (TSG) activation and its biological role in oral squamous cell carcinoma (OSCC) remain unknown. Here, we report procaine inhibited DNA methylation by suppressing DNMT activity and increased the expression of PAX9, a differentiation gene in OSCC cells. Interestingly, the reactivation of PAX9 by procaine found to inhibit cell growth and trigger apoptosis in OSCC in vitro and in vivo. Likely, the enhanced PAX9 expression after exposure to procaine controls stemness and differentiation through the autophagy-dependent pathway in OSCC cells. PAX9 inhibition abrogated procaine-induced apoptosis, autophagy, and inhibition of stemness. In OSCC cells, procaine improved anticancer drug sensitivity through PAX9, and its deficiency significantly blunted the anticancer drug sensitivity mediated by procaine. Additionally, NRF2 activation by procaine facilitated the antitumor response of PAX9, and pharmacological inhibition of NRF2 by ML385 reduced death and prevented the decrease in the orosphere-forming potential of OSCC cells. Furthermore, procaine promoted antitumor activity in FaDu xenografts in athymic nude mice, and immunohistochemistry data showed that PAX9 expression was significantly enhanced in the procaine group compared to the vehicle control. In conclusion, PAX9 reactivation in response to DNMT inhibition could trigger a potent antitumor mechanism to provide a new therapeutic strategy for OSCC.     

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone,a polymethoxyflavone,exerts antitumor effect on PI3K/Akt signaling pathway in human gastric cancer cell BGC-7901

5-Hydroxy-3,6,7,8,3′,4′-hexamethoxyflavone (5HHMF), a polymethoxyflavone (PMF) mainly found in citrus plants, exhibits excellent physiological functions. In this study, we aimed to investigate the anticancer activity of 5HHMF against human gastric cancer cell BGC-7901 both in vitro and in vivo and illustrate the potential mechanisms. The proliferation of BGC-7901 cells was assessed by MTT assay. Reactive oxygen species (ROS) level was determined by ELISA kit. The protein expression was determined by western blot analysis. Antitumor activity of 5HHMF in vivo was evaluated in BALB/c nude mice. The results showed that treatment with 5HHMF significantly suppressed BGC-7901 cells proliferation, increased ROS generation, and upregulated cytochrome c release from the mitochondria to the cytosol. Western blot analysis demonstrated that 5HHMF significantly downregulated the expression of procaspase-3, procaspase-9, and PARP and upregulated cleaved caspase-3, cleaved caspase-9, cleaved PARP, and Bax/Bcl-2 ratio. Meanwhile, 5HHMF treatment markedly decreased the expression of PI3K and p-Akt. In addition, 5HHMF effectively inhibited tumor growth in xenograft models in BALB/c nude mice without major side action. In summary, 5HHMF-induced apoptosis via targeting PI3K/Akt, indicating 5HHMF is a potential antitumor agent for gastric cancer.