Babao Dan decreases hepatocarcinogenesis by inhibiting hepatic progenitor cells malignant transformation via down-regulating toll-like receptor 4.

Background: Babao Dan (BBD) is a traditional Chinese medicine that has been widely used as a complementary and alternative medicine to treat chronic liver diseases. In this study, we aimed to observe the effect of BBD on the incidence of diethylnitrosamine (DEN)-initiated hepatocellular carcinoma formation in rats and explored its possible mechanism. Methods: To verify this hypothesis, BBD was administrated to rats at a dose of 0.5g/kg body weight per two days from the 9th to 12th week in HCC-induced by DEN. Liver injury biomarkers and hepatic inflammatory parameters were evaluated by histopathology as well as serum and hepatic content analysis. We applied immunohistochemical analysis to investigate the expression of CK-19 and SOX-9 in liver tissues. The expression of TLR4 was determined by immunohistochemical, RT-PCR, and western blot analysis. Furthermore, we also detected the efficacy of BBD against primary HPCs neoplastic transformation induced by LPS. Results: We observed that DEN could induce hepatocarcinogenesis, and BBD could obviously decrease the incidence. The biochemical and histopathological examination results confirmed that BBD could protect against liver injury and decrease inflammatory infiltration. Immunohistochemistry staining results showed that BBD could effectively inhibit the ductal reaction and the expression of TLR4. The results showed that BBD-serumcould obviously inhibit primary HPCs neoplastic transformation induced by regulating the TLR4/Ras/ERK signaling pathway. Conclusion: In summary, our results indicate that BBD has potential applications in the prevention and treatment of HCC, which may be related to its effect on hepatic progenitor cells malignant transformation via inhibiting the TLR4/Ras/ERK signaling pathway.

Lipopolysaccharide induces the differentiation of hepatic progenitor cells into myofibroblasts constitutes the hepatocarcinogenesis-associated microenvironment.

Hepatocellular carcinoma (HCC) generally occurs in the presence of chronic liver injury, often as a sequela of liver fibrosis. Hepatic progenitor cells (HPCs) are known to be capable of forming both hepatocytes and cholangiocytes in chronic liver injury, which are also considered a source of myofibroblasts and tumor-initiating cells, under carcinogenic circumstances. However, the underlying mechanisms that activate HPCs to give rise to HCC are still unclear. In current study, the correlation between HPCs activation and liver fibrosis and carcinogenesis was investigated in rats and human specimens. We analyzed the role of HPCs in tumorigenesis, by transplanting exogenous HPCs in a diethylnitrosamine-induced rat HCC model. Our data indicated that HPC activation correlated with hepatic fibrosis and hepatocarcinogenesis. We further found that exogenous HPC infusion promoted liver fibrosis and hepatocarcinogenesis, while lipopolysaccharides (LPS) played an important role in this process. However, results of our study indicated that LPS did not induce HPCs to form tumor in nude mice directly. Rather, LPS induced myofibroblast-like morphology in HPCs, which enhanced the tumorigenic potential of HPCs. Further experiments showed that LPS/Toll-like receptor 4 (TLR4) signaling mediated the differentiation of HPCs into myofibroblasts and enhanced the production of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), which led to the aberrant expression of Ras and p53 signaling pathways in HPCs, and finally, promoted the proliferation and malignant transformation of HPCs, by long non-coding RNA regulation. Besides, examination of HCC clinical samples demonstrated that IL-6 and TNF-α production correlated with HPC activation, hepatic fibrosis, and HCC recurrence. Our study indicates that both myofibroblasts and tumor cells are derived from HPCs. HPC-derived myofibroblasts create tumor microenvironment and contribute to the proliferation and malignant transformation of HPCs. Furthermore, LPS present in the chronic liver inflammation microenvironment might play an important role in hepatocarcinogenesis, by regulating the plastic potential of HPCs.

Look into hepatic progenitor cell associated trait: Histological heterogeneity of hepatitis B-related combined hepatocellular-cholangiocarcinoma.

Combined hepatocellular-cholangiocarcinoma (CHC) is a mixed tumor containing elements of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC). Its remarkable histological heterogeneity has been linked to putative hepatic progenitor cell (HPC) origin. However, detailed histological or phenotypic description is rarely documented. In the present study, we reassessed 68 cases previously diagnosed as hepatitis B-related CHCs by immunohistochemistry and double-fluorescence immunostaining, focusing on HPC associated phenotypic observation of intermediate area of the tumor. It was found that tumor cells showed remarkable heterogeneity in intermediate area. Tumor cells with intermediate morphology between hepatocytes and cholangiocytes were oval-shaped and small with scant cytoplasm and hyperchromatic nuclei, arranging in solid nests mostly. By Keratin 7 (K7) staining, it appeared that the nests of tumor cells represented a maturation process from the undifferentiated small cells to mature hepatocytes through the "transitional" cells. Then, these small cells were further confirmed with intermediate phenotype as HPC by exploring immature hepatocellular marker and HPC/biliary markers co-localization. In conclusion, the HPC associated trait in CHC can be interpreted by HPC origin or gain of "stemness" by dedifferentiation. It is still too soon to give a final word that it is innate or acquired signature of HPC associated trait in CHC.

Methylation mediated Gadd45ß enhanced the chemosensitivity of hepatocellular carcinoma by inhibiting the stemness of liver cancer cells.

BACKGROUND: Defects of the growth arrest DNA damage-inducible gene 45ß (Gadd45ß) play an important role in the progression of tumor and confer resistance to chemotherapy. However, the role of Gadd45ß in the apoptosis of hepatocellular carcinoma is still not clear. Purpose of this study was to explore the effect of Gadd45ß on the apoptosis of liver cancer cells, and the possible mechanism was examined. RESULT: In this study, we first confirmed the decreased expression of Gadd45ß in human liver cancer tissues and human liver cancer cell lines, when compared to the peri-tumor liver tissue and normal liver cells. And, it was found that Gadd45ß could inhibit the stemness of liver cancer cells, enhancing the apoptosis of cancer cells induced by chemotherapy. Furthermore, the results showed that HCC tissues and cell lines showed a higher methylation status in Gadd45ß promoter than that in peri-tumor tissues and normal liver cells. Methylation was then reversed by pretreatment of SMMC-7721 and Hep-3B with 5-azacytidine which is the DNA methyltransferase inhibitor. And the 5-azacytidine decreased the stemness of SMMC-7721 and Hep-3B, enhanced the sensitivity of SMMC-7721 and Hep-3B to cisplatin. CONCLUSIONS: Methylation mediated Gadd45ß expression inhibited the stemness of liver cancer cells, promoting the chemotherapy-induced apoptosis. Thus Gadd45ß may be the potential target for enhancing the chemosensitivity of human hepatocellular carcinoma.

Suppression of p53 potentiates chemosensitivity in nutrient-deprived cholangiocarcinoma cells via inhibition of autophagy.

Tumor protein p53 has been intensively studied as a major tumor suppressor. The activation of p53 is associated with various anti-neoplastic functions, including cell senescence, cell cycle arrest, apoptosis and inhibition of angiogenesis. However, the role of p53 in cancer cell chemosensitivity remains unknown. Cholangiocarcinoma cell lines QBC939 and RBE were grown in full-nutrient and nutrient-deprived conditions. The cell lines were treated with 5-fluorouracil or cisplatin and the rate of cell death was determined in these and controls using Cell Counting Kit-8 and microscopy-based methods, including in the presence of autophagy inhibitor 3MA, p53 inhibitor PFT-α or siRNA against p53 or Beclin-1. The present study demonstrated that the inhibition of p53 enhanced the sensitivity to chemotherapeutic agents in nutrient-deprived cholangiocarcinoma cells. Nutrient deprivation-induced autophagy was revealed to be inhibited following inhibition of p53. These data indicate that p53 is important for the activation of autophagy in nutrient-deprived cholangiocarcinoma cells, and thus contributes to cell survival and chemoresistance.

LPS-induced CXCR4-dependent migratory properties and a mesenchymal-like phenotype of colorectal cancer cells.

Colorectal cancer (CRC) is the most commonly diagnosed cancer worldwide, and over 50% of patients will develop hepatic metastasis during the course of their disease. CXCR4 and its ligand, stromal cell-derived factor 1α (SDF-1α)/chemokine (C-X-C motif) ligand 12 (CXCL12) have been revealed as regulatory molecules involved in the spreading and progression of a variety of tumors. Here we have shown that lipopolysaccharides (LPS) promoted the migratory capacity of colon cancer cells in vivo and in vitro, which correlated with the activation of SDF-1α/CXCR4 axis and epithelial-mesenchymal transition (EMT) occurrence. Additionally, we found that LPS-induced CXCR4 expression and EMT through NF-κB signaling pathway activation. And inhibition of NF-κB pathway, which recovered the epithelial phenotype and attenuated CXCR4 expression, inhibited cell migratory capacity. Clinically, high levels of CXCR4 always correlated with metastasis and poor prognosis of CRC patients. In conclusion, LPS participate in the whole process of hepatic metastasis of CRC, not only causing liver damage resulting in the production of SDF-1α, but also enhancing the invasive potential of CRC cells by promoting CXCR4 expression and EMT occurrence, which would contribute to the enhancement of cell migration and invasion.

Lipopolysaccharide promotes tumorigenicity of hepatic progenitor cells by promoting proliferation and blocking normal differentiation.

Hepatic progenitor cells (HPCs) are bipotential stem cells that can differentiate into mature hepatocytes or biliary epithelial cells (BECs). They are thought to be involved in repair of liver injury and the incidence of hepatic carcinoma. Their physiology is closely associated with the microenvironment where they reside. Lipopolysaccharide (LPS), an important component of the hepatic pathological microenvironment, is stored in the liver and affects many types of cells in various hepatosis. HPCs may also be influenced by LPS. In this paper, mouse ED13.5 E-cadherin+ foetal liver cells were isolated as mouse hepatic progenitor cells (mHPCs). Proliferation of mHPCs was promoted under LPS conditions both in vivo and in vitro. Moreover, LPS enhanced colony formation ability of mHPCs, and blocked them differentiation into mature hepatocytes and formation of a bile duct-liked structure. More importantly, long-term treatment with LPS promoted tumorigenesis of mHPCs in nude mice. Thus, we conclude that LPS may promote aberrant proliferation of mHPCs and restrict their normal differentiation. Long-term exposure of mHPCs to LPS increased the risk of tumour formation. These data provide insight into the links between LPS, HPCs fate, and tumorigenesis, and present novel insight into the relationship between HPCs and their microenvironment.

Look into Hepatic Progenitor Cell Associated Trait: Histological Heterogeneity of Hepatitis B-Related Combined Hepatocellular-Cholangiocarcinoma / 华中科技大学学报（医学）（英德文版）

Combined hepatocellular-cholangiocarcinoma (CHC) is a mixed tumor containing elements of both hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC).Its remarkable histological heterogeneity has been linked to putative hepatic progenitor cell (HPC) origin.However,detailed histological or phenotypic description is rarely documented.In the present study,we reassessed 68 cases previously diagnosed as hepatitis B-related CHCs by immunohistochemistry and double-fluorescence immunostaining,focusing on HPC associated phenotypic observation of intermediate area of the tumor.It was found that tumor cells showed remarkable heterogeneity in intermediate area.Tumor cells with intermediate morphology between hepatocytes and cholangiocytes were oval-shaped and small with scant cytoplasm and hyperchromatic nuclei,arranging in solid nests mostly.By Keratin 7 (K7) staining,it appeared that the nests of tumor cells represented a maturation process from the undifferentiated small cells to mature hepatocytes through the "transitional" cells.Then,these small cells were further confirmed with intermediate phenotype as HPC by exploring immature hepatocellular marker and HPC/biliary markers co-localization.In conclusion,the HPC associated trait in CHC can be interpreted by HPC origin or gain of"stemness" by dedifferentiation.It is still too soon to give a final word that it is innate or acquired signature of HPC associated trait in CHC.

Lipopolysaccharide supports maintaining the stemness of CD133(+) hepatoma cells through activation of the NF-κB/HIF-1α pathway.

Due to the existence of cancer stem cells (CSCs), persistence and relapse of human hepatocellular carcinoma (HCC) are common after treatment with existing anti-cancer therapies. Emerging evidence indicates that lipopolysaccharide (LPS) plays a crucial role in aggravating HCC, but information about the effect of LPS on CSCs of HCC remains scant. Here, we report that the stemness of CD133(+) CSCs sorted from the human HCC cell line Huh7 was maintained well when cells were cultured with LPS. The reduction of CD133 expression was much lesser in cultured CSCs in the presence of LPS. In response to LPS stimulation, CSCs showed an increase in their activity of clonogenesis and tumorigenesis. LPS also supported maintaining CSC abilities of migration, invasion, and chemo-resistance. Treatment with HIF-1α-specific siRNA significantly reduced CD133 expression by CSCs at both mRNA and protein levels. Further, the expression of HIF-1α and CD133 was reduced in LPS-stimulated CSCs when the NF-κB inhibitor was added to the cell culture. HIF-1α-specific siRNA also effectively counteracted the effect of LPS on maintaining CSC abilities of migration and invasion. These data indicate that LPS, an important mediator in the liver tumor microenvironment, supports the maintenance of CSC stemness through signaling of the NF-κB/HIF-1α pathway. Our current study highlights LPS as a potential target for developing new therapeutic approaches to eliminate CSCs during the treatment of HCC.

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53.

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.

Hepatitis B virus (HBV) receptors: Deficiency in tumor results in scant HBV infection and overexpression in peritumor leads to higher recurrence risk.

Hepatitis B virus (HBV) infection is a risk factor for hepatocarcinogenesis and recurrence. Here, we sought to characterize intratumoral and peritumoral expression of HBsAg and its specific receptors in HBsAg-positive hepatocellular carcinoma (HCC) patients and further examined their correlation with the recurrence-free survival (RFS). HCC tissue and adjacent normal tissue specimens were acquired from HBsAg-positive patients. The presence of HBsAg and receptors, as well as hepatic progenitor cells (HPCs) were detected by tissue microassay and immunohistochemistry. Necroinflammatory activity was evaluated by HE staining. The mean IOD of HBsAg and HBV DNA in the intratumoral tissues was markedly lower than that in the peritumoral tissues (P < 0.001). Pearson correlation analysis further showed a significant correlation between the expression of HBsAg and NTCP (r = 0.461, P < 0.001) or ASGPR (r = 0.506, P < 0.001) in peritumoral tissues. And the peritumoral HBsAg and receptors presented a positive association with necroinflammatory activity (P < 0.05). Inflammation induced by HBV infection presented a positive association with HPCs activation (P < 0.05). Additionally, due to lack of HBV receptors, HPCs was not preferentially infected with HBV, but activated HPCs had a significant correlation with HBsAg expression in peritumoral tissues, and the peritumoral HPCs activation was associated with RFS of HCC patients, therefore, the overexpression of HBsAg and receptors in peritumor were also with higher recurrence risk (P < 0.05). In conclusion, lack of HBV receptors resulted in scant HBV infection in tumor cells, and overexpression of HBsAg and receptors in peritumor was strongly associated with higher recurrence risk in HCC patients.

The injured liver induces hyperimmunoglobulinemia by failing to dispose of antigens and endotoxins in the portal system.

Hyperimmunoglobulinemia is frequently observed in patients with chronic liver diseases. However, the exact mechanism underlying the high level of antibody formation is not fully understood. In our study, we provide evidence for the functional role of the liver and the stimulation of plasma cell proliferation in hyperimmunoglobulinemia. We collected sera from patients with chronic liver diseases, and the level of serum immunoglobulins in patients was examined; this was also investigated in animal models of liver cirrhosis and hepatocellular carcinoma. An end-to-side microsurgical portacaval shunt was used to mimic liver dysfunction in rats. We used portal vein serum and inferior vena cava serum to immunize healthy rats and mice in order to confirm the function of the healthy liver in disposing of antigens and endotoxins from the gut. For the analysis of the state of plasma cell activation, plasma cells from mice were stained with PE-conjugated anti-CD138 and FITC-conjugated anti-BrdU for flow cytometry analysis. Hyperimmunoglobulinemia was observed both in patients with chronic liver diseases and in related animal models, and high plasma LPS levels were also observed. There was a significant increase in the activation and proliferation of plasma cell in mice immunized with antigens or LPS-positive serum compared with controls that were immunized with antigens and LPS-negative serum. We confirmed that the healthy liver plays an important role in disposing of antigens and endotoxins derived from the gut. Hyperimmunoglobulinemia in chronic liver diseases mainly arises due to the collateral circulation secondary to portal hypertension, gut antigens and endotoxins that bypass the liver and reach the antibody-producing cells.

Toll like receptor 4 facilitates invasion and migration as a cancer stem cell marker in hepatocellular carcinoma.

Cancer stem cells (CSCs) or tumor-initiating cells (TICs), a small subset of tumor cells, are involved in tumor initiation, progression, recurrence and metastasis. In human hepatocellular carcinoma (HCC), TICs are enriched with cell surface markers and play a key role in chemotherapy resistance, tumor invasion and migration. Toll like receptor 4 (TLR4), acting as a receptor for lipopolysaccharide (LPS), has been reported to be responsible for carcinogenesis, invasion, metastasis and cancer progression. In our study, two HCC cell lines and a splenic vein metastasis of the nude mouse model were used to study the invasive ability of TLR4 positive HCC cells in vitro and in vivo. Stem-like features were also detected in TLR4 positive HCC cells. A total of 88 clinical samples from HCC patients were used to evaluate the association of TLR4 and stem-cell marker expression, and the relationship between TLR4 expression and clinicopathological characteristics was analyzed. The in vitro and in vivo experiments indicated that TLR4 positive HCC cells displayed significantly enhanced invasion and migration, and stem-like properties were also detected in TLR4 positive HCC cells. Clinically, TLR4 expression levels were found to be significantly higher in HCC tissues with microvascular invasion. Additionally, high expression of TLR4 in HCC tissues was strongly associated with both early recurrence and poor survivals in patients. Our results indicated that there was a relationship between TLR4 expression and CSC's features, TLR4 may act as a CSC marker, prompting tumor invasion and migration, which contributes to the poor prognosis of HCC.

Overexpression of SIRT1 promotes metastasis through epithelial-mesenchymal transition in hepatocellular carcinoma.

BACKGROUND: SIRT1 is a member of the mammalian sirtuin family with the ability to deacetylate histone and nonhistone proteins. The correlation between SIRT1 expression and tumor metastasis in several types of cancer has aroused widespread concern. This study investigated SIRT1 expression and its prognostic value in hepatocellular carcinoma (HCC). The function of SIRT1 in hepatocarcinogenesis was further investigated in cell culture and mouse models. METHODS: Western blotting and immunohistochemistry were used to explore SIRT1 expression in HCC cell lines and primary HCC clinical specimens. The functions of SIRT1 in the migration and invasion in the HCC cell line were analyzed by infecting cells with adenovirus containing full-length SIRT1 or sh-RNA. The effect of SIRT1 on tumorigenicity in nude mice was also investigated. RESULTS: SIRT1 expression was significantly overexpressed in the tumor tissues and HCC cell lines. SIRT1 significantly promoted the ability of migration and invasion in HCC cells. In addition, experiments with a mouse model revealed that SIRT1 overexpression enhanced HCC tumor metastasis in vivo. Furthermore, we demonstrated that SIRT1 significantly enhanced the invasive and metastatic potential by inducing epithelial-mesenchymal transition in HCC cells. A clinicopathological analysis showed that SIRT1 expression was significantly correlated with tumor size, tumor number, and TNM staging. Kaplan-Meier survival curves revealed that positive SIRT1 expression was associated with poor prognosis in patients with HCC. CONCLUSIONS: Our data suggest that SIRT1 may play an important role in HCC progression and could be a potential molecular therapy target for HCC.

Tumor-associated macrophages promote cancer stem cell-like properties via transforming growth factor-beta1-induced epithelial-mesenchymal transition in hepatocellular carcinoma.

Tumor-associated macrophages (TAMs), a crucial component of immune cells infiltrated in tumor microenvironment, have been found to be associated with progression and metastasis of hepatocellular carcinoma (HCC). In this study, we aimed to clarify the mechanism underlying the crosstalk between TAMs and cancer stem cells (CSCs) in HCC. Mouse macrophage cell line RAW264.7 cells were used to investigate the effects of TAMs on mouse hepatoma cell line Hepa1-6 cells in vivo and vitro. A total of 90 clinical samples had pathology-proven HCC were used to evaluate the distribution of TAMs and CSCs and analyze their value in predicting the prognosis. In the study, we have found that the number of TAMs has a positive correlation with the density of CSCs in the marginal of human HCC. Our results show that, cocultured with TAM-conditioned medium (CM) promoted CSC-like properties in Hepa1-6 cells, which underwent EMT and gained higher invasive capability. TAMs secreted more transforming growth factor- beta1 (TGF-beta1) than other phenotypes of macrophage. Furthermore, depletion of TGF-beta1 blocked acquisition of CSC-like properties by inhibition of TGF-beta1-induced EMT. High expression of CD68 in the EpCAM positive expression HCC tissues was strongly associated with both poor cancer-free survival and overall survival in patients. Our results indicate that the TAMs promote CSC-like properties via TGF-beta1-induced EMT and they may contribute to investigate the prognosis of HCC.

Autophagy inhibition switches low-dose camptothecin-induced premature senescence to apoptosis in human colorectal cancer cells.

Recently, several studies indicated that senescent tumor cells are resistant to apoptosis in chemotherapy. They may return to cell cycle, thus act as stumbling blocks in anticancer treatments. In the present study, we found that, in human colorectal cancer cells, low-dose camptothecin (CPT) simultaneously induced autophagy and premature senescence through AMPK-TSC2-mTOR pathway and ATM-Chk2-p53-p21 pathway respectively. What's important is the suppression of autophagy substantially increased apoptosis and greatly attenuated senescence possibly by blocking p53/p21 pathway, which suggests that autophagy plays an indispensable role in sustaining cell senescence caused by low-dose CPT. The combination of low-dose CPT and autophagy inhibitor, a way to lead senescent cells to die, would be potentially valuable in cancer therapy.

Inhibition of p53 increases chemosensitivity to 5-FU in nutrient-deprived hepatocarcinoma cells by suppressing autophagy.

Activation of p53 can induce apoptosis, cell cycle arrest, and cell senescence, although some evidence has suggested that p53 could promote cell survival. However, whether p53 plays a positive role in cancer cell survival to chemotherapy remains unknown. In this study, we show that inhibition of p53 enhanced apoptosis and increased chemosensitivity to 5-fluorouracil (5-FU) in nutrient-deprived hepatocarcinoma cells (HCC). Meanwhile, nutrient-deprivation-induced autophagy was inhibited by pifithrin-α or small interfering RNA targeting p53. The expression of p53 was not increased when HCC were incubated under nutrient-deprived conditions. This indicates that the basal level of p53 is important to autophagy activation in nutrient-deprived HCC cells. Furthermore, combining p53 inhibition and nutrient deprivation or 5-FU treatment resulted in a marked increase in reactive oxygen species generation and mitochondrial damage. Antioxidants reduced nutrient deprivation or 5-FU-induced cell death of HCC after p53 inhibition. Our results suggest that p53 contributes to cell survival and chemoresistance in HCC under nutrient-deprived conditions by modulating autophagy activation.

Autophagy contributes to the survival of CD133+ liver cancer stem cells in the hypoxic and nutrient-deprived tumor microenvironment.

Liver cancer stem cells (LCSCs) can drive and maintain hepatocellular carcinoma (HCC) growth, metastasis, and recurrence. Therefore, they are potentially responsible for the poor prognosis of HCC. Oxygen and nutrient deficiencies are common characteristics of the tumor microenvironment. However, how LCSCs adapt to oxygen- and nutrient-deprived conditions is unclear. Here, we used immunofluorescent staining and flow cytometry analysis to show that CD133+ cells were significantly enriched after hypoxia and nutrient starvation (H/S) in the human HCC cell line Huh7. Sorted CD133+ cells showed higher survival, less apoptosis, and possess higher clonogenic ability under H/S compared to the CD133- population. Under H/S, electron microscopy revealed more advanced autophagic vesicles in CD133+ cells. Additionally, CD133+ cells had higher autophagy levels as measured by both RT-qPCR and Western blotting. CD133+ cells had more accumulated GFP-LC3 puncta, which can be detected by fluorescence microscopy. The autophagic inhibitor chloroquine (CQ) significantly increased apoptosis and decreased the clonogenic capacity of CD133+ cells under H/S. Pre-culturing in H/S enhanced the sphere-forming capacity of CD133+ cells. However, CQ significantly impaired this process. Therefore, autophagy is essential for LCSCs maintenance. CD133+ cells were also found to have a higher tumor-forming ability in vivo, which could be inhibited by CQ administration. Collectively, our results indicate that the involvement of autophagy in maintenance of CD133+ LCSCs under the oxygen- and nutrient-deprived conditions that are typical of the tumor microenvironment in HCC. Therefore, autophagy inhibitors may make LCSCs more sensitive to the tumor microenvironment and be useful in improving anti-cancer treatments.

Chloroquine promotes the anticancer effect of TACE in a rabbit VX2 liver tumor model.

BACKGROUND: To investigate the efficacy of TACE combined with CQ, an autophagic inhibitor, in a rabbit VX2 liver tumor model. METHODS: Tumor size was measured. And tumor growth rate was calculated to examine the effect of the combined treatment. Apoptosis was detected by TUNEL assay. Meanwhile, autophagic activity was detected by immunohistochemistry and Western blotting to investigate the mechanism underlying. Liver function was also examined to assess feasibility and safety of the combined therapy. RESULTS: Tumors in the control grew more than 4 times bigger after 14 days, while that in the group of TACE alone just showed mild growth. But a slight shrinkage was shown after the treatment of CQ+TACE. Growth ratio of TACE alone was 96.45% ± 28.958% while that of CQ+TACE was -28.73% ± 12.265%. Compared with TACE alone, necrosis in CQ+TACE showed no significant difference, however, the apoptosis was much higher. There were only 14.8±3.11% apoptotic cells in TACE, but 33±4.18% in CQ+TACE, which suggests the increased apoptosis in CQ+TACE contributed to the decrease of tumor volume. In terms of autophagic activity, the result is negative when we immunostained sections of the control with LC3 antibody, but positive in TACE alone and CQ+TACE. And the result of Western blot showed that there was just a low level of LC3Ⅱexpressed in the control and CQ alone, but higher in TACE, and much higher in CQ+TACE because CQ inhibited its degradation in autophagy. Compared with control, p62 decreased in TACE, but the decrease was partially reversed in CQ+TACE. In addition, toxicity of CQ+TACE was assessed not higher than TACE alone, which supports the safety of CQ+TACE. CONCLUSION: CQ+TACE works better than TACE alone in rabbit VX2 liver tumor model because CQ inhibits autophagy induced by TACE. The inhibited autophagy loses its resistance to apoptosis that apoptosis increased, which contributes to the inhibition of tumor growth. This study indicates CQ may be a promising adjuvant to promote the effect of TACE.

Inhibition of autophagy enhances anticancer effects of bevacizumab in hepatocarcinoma.

Angiogenesis inhibitors have long been considered desirable anticancer agents. However, it was found that many tumors could develop resistance to antiangiogenesis inhibitors. Antiangiogenic therapy results in metabolic stress. Autophagy is an important survival mechanism in cancer cells under metabolic stress; however, it remains unknown if autophagy contributes to antiangiogenesis resistance. In this study, we reported that bevacizumab treatment reduced the development of new blood vessels and inhibited cell growth in xenografts of hepatocellular carcinoma (HCC) tumors. Bevacizumab treatment also upregulated expression of the autophagy-related genes (Beclin1 and LC3) and increased autophagosome formation. Our in vitro studies demonstrated that autophagy inhibition significantly increased apoptosis of HCC cells during nutrient starvation or hypoxia. In addition, the combined treatment of an autophagy inhibitor and bevacizumab markedly inhibited the tumor growth of HCC xenografts, led to enhanced apoptosis, and impaired the proliferation of tumor cells compared with treatment with either drug alone. Furthermore, autophagy inhibition led to enhanced reactive oxygen species (ROS) generation in HCC cells exposed to nutrient starvation or hypoxia in vitro and increased DNA oxidative damage in vivo. Antioxidants reduced nutrient starvation or the hypoxia-induced cell death of HCC cells after autophagy inhibition. Our results suggest that autophagy modulates ROS generation and contributes to cell survival under metabolic stress. Therefore, autophagy inhibition may be a novel way of increasing the efficicacy of antiangiogenic agents in the treatment of HCC.