Terence Kin-Wah Lee

Aldehyde Dehydrogenase Discriminates the CD133 Liver Cancer Stem Cell Populations

Recent efforts in our study of cancer stem cells (CSC) in hepatocellular carcinoma (HCC) have led to the identification of CD133 as a prominent HCC CSC marker. Findings were based on experiments done on cell lines and xenograft tumors where expression of CD133 was detected at levels as high as 65%. Based on the CSC theory, CSCs are believed to represent only a minority number of the tumor mass. This is indicative that our previously characterized CD133+ HCC CSC population is still heterogeneous, consisting of perhaps subsets of cells with differing tumorigenic potential. We hypothesized that it is possible to further enrich the CSC population by means of additional differentially expressed markers. Using a two-dimensional PAGE approach, we compared protein profiles between CD133+ and CD133− subpopulations isolated from Huh7 and PLC8024 and identified aldehyde dehydrogenase 1A1 as one of the proteins that are preferentially expressed in the CD133+ subfraction. Analysis of the expression of several different ALDH isoforms and ALDH enzymatic activity in liver cell lines found ALDH to be positively correlated with CD133 expression. Dual-color flow cytometry analysis found the majority of ALDH+ to be CD133+, yet not all CD133+ HCC cells were ALDH+. Subsequent studies on purified subpopulations found CD133+ALDH+ cells to be significantly more tumorigenic than their CD133−ALDH+ or CD133−ALDH− counterparts, both in vitro and in vivo. These data, combined with those from our previous work, reveal the existence of a hierarchical organization in HCC bearing tumorigenic potential in the order of CD133+ALDH+ > CD133+ALDH− > CD133−ALDH−. ALDH, expressed along CD133, can more specifically characterize the tumorigenic liver CSC population. (Mol Cancer Res 2008;6(7):1146–53)

Graft injury in relation to graft size in right lobe live donor liver transplantation: a study of hepatic sinusoidal injury in correlation with portal hemodynamics and intragraft gene expression.

ObjectiveTo investigate the degree and mechanism of hepatic sinusoidal injury in different graft sizes in right lobe live donor liver transplantation (LDLT). Summary Background DataLiver grafts from living donors are likely to be small-for-size for adult recipients. Graft injury after reperfusion is common, but the mechanism and degree of injury remain unclear. The hepatic sinusoidal injury in different graft sizes and its relationship with portal hemodynamics and intragraft gene response at the early phase after reperfusion have not been studied in right lobe LDLT. MethodsFrom May 2000 to November 2001, 40 adults receiving right lobe LDLT had portal pressure measured continuously before and after reperfusion. Liver biopsies were taken before and after reperfusion for detection of vasoregulatory genes (endothelin-1 and endothelial nitric oxide synthase) and heat shock genes (heat shock protein 70 and heme oxygenase-1), and electron microscope examination. Blood samples from the portal vein and suprahepatic inferior vena cava were taken for the measurement of plasma nitric oxide level. ResultsThe recipients were grouped according to the ratio of graft weight to estimated standard liver weight: group 1 (n = 10), less than 40%; group 2 (n = 21), 40% to 60%; and group 3 (n = 9), more than 60%. The portal pressures recorded after reperfusion in group 1 were significantly higher within 30 minutes of reperfusion than those in groups 2 and 3. After reperfusion, the intragraft endothelin-1 mRNA level in group 1 increased by 161% of the basal level but decreased by 31.5% and 62% of the basal level in groups 2 and 3, respectively. The intragraft mRNA level of heme oxygenase-1 in groups 1 and 2 decreased by 75.5% and 25.3% of the basal level respectively but increased by 41% of basal level in group 3. The intragraft protein level of heat shock protein 70 decreased by 50 ng/mL after reperfusion in group 1 but increased by 12.4 ng/mL and 0.6 ng/mL in groups 2 and 3, respectively. The portal vein plasma nitric oxide level decreased more significantly after reperfusion in group 1 than in group 2. Electron microscope examination of liver biopsies in group 1 showed tremendous mitochondrial swelling as well as irregular large gaps between the sinusoidal lining cells. There were two hospital deaths in group 1 and none in the other two groups. ConclusionsPatients implanted with grafts less than 40% of standard liver weight suffered from transient portal hypertension early after reperfusion. The phenomenon was accompanied by intragraft upregulation of endothelin-1 and ultrastructural evidence of sinusoidal damage. The transient portal hypertension after reperfusion, subsequent endothelin-1 overexpression, and plasma nitric oxide level reduction, together with downregulation of heme oxygenase-1 and heat shock protein 70, may account for the small-for-size graft injury.

Gamma-tocotrienol as an effective agent in targeting prostate cancer stem cell-like population.

Emerging evidence supports that prostate cancer originates from a rare subpopulation of cells, namely prostate cancer stem cells (CSCs). Conventional therapies for prostate cancer are believed to mainly target the majority of differentiated tumor cells but spare CSCs, which may account for the subsequent disease relapse after treatment. Therefore, successful elimination of CSCs may be an effective strategy to achieve complete remission from this disease. Gamma‐tocotrienols (γ‐T3) is one of the vitamin‐E constituents, which have been shown to have anticancer effects against a wide range of human cancers. Recently, we have reported that γ‐T3 treatment not only inhibits prostate cancer cell invasion but also sensitizes the cells to docetaxel‐induced apoptosis, suggesting that γ‐T3 may be an effective therapeutic agent against advanced stage prostate cancer. Here, we demonstrate for the first time that γ‐T3 can downregulate the expression of prostate CSC markers (CD133/CD44) in androgen‐independent prostate cancer cell lines (PC‐3 and DU145), as evident from Western blotting analysis. Meanwhile, the spheroid formation ability of the prostate cancer cells was significantly hampered by γ‐T3 treatment. In addition, pretreatment of PC‐3 cells with γ‐T3 was found to suppress tumor initiation ability of the cells. More importantly, although CD133‐enriched PC‐3 cells were highly resistant to docetaxel treatment, these cells were as sensitive to γ‐T3 treatment as the CD133‐depleted population. Our data suggest that γ‐T3 may be an effective agent in targeting prostate CSCs, which may account for its anticancer and chemosensitizing effects reported in previous studies.

Distinct intragraft response pattern in relation to graft size in liver transplantation.

Background. The molecular mechanism of small-for-size graft injury remains unclear. The aim of this study is to investigate the gene expression pattern of acute phase response in relation to graft size in a rat-liver transplantation model. Methods. A rat orthotopic liver transplantation model using 30%, 50%, and whole grafts was used. The graft survival rates and liver morphology were compared among the three groups. Two transcription factors, nuclear factor (NF)-&kgr;B (p65) and early growth response (Egr-1), and their downstream genes were compared. Results. According to the graft size, the rats were grouped as follows: group 1 (n=20), 32% (24–47%); group 2 (n=10), 56% (50–65%); and group 3 (n=10), 104% (89–120%). The 7-day survival rates were 20% (P =0.039 vs. group 2, P =0.000 vs. group 3), 60%, and 100% in groups 1, 2, and 3, respectively. Dilation of hepatic sinusoids and vacuolization of hepatocytes were observed in group 1. Up-regulation of Egr-1 and endothelin (ET)-1 and over-expression of nitric oxide synthase (iNOS) was found in group 1, but heme oxygenase (HO)-1 and A20 were down-regulated. At 24 hours after reperfusion, the intragraft protein level of heat-shock protein (Hsp)-70 was significantly lower in group 1 than that in group 3 (12.4 vs. 17.0 ng/mL, P =0.04). More apoptotic nuclei were found in group 1. Conclusions. Small-for-size graft injury was related to early over-expression of Egr-1 associated with up-regulation of ET-1 and deterioration of intracellular homeostasis reflected by down-regulation of Hsps and A20.

Lupeol targets liver tumor-initiating cells through phosphatase and tensin homolog modulation†

Liver tumor‐initiating cells (T‐ICs) are capable of self‐renewal and tumor initiation and are more chemoresistant to chemotherapeutic drugs. The current therapeutic strategies for targeting stem cell self‐renewal pathways therefore represent rational approaches for cancer prevention and treatment. In the present study, we found that Lup‐20(29)‐en‐3β‐ol (lupeol), a triterpene found in fruits and vegetables, inhibited the self‐renewal ability of liver T‐ICs present in both hepatocellular carcinoma (HCC) cell lines and clinical HCC samples, as reflected by hepatosphere formation. Furthermore, lupeol inhibited in vivo tumorigenicity in nude mice and down‐regulated CD133 expression, which was previously shown to be a T‐IC marker for HCC. In addition, lupeol sensitized HCC cells to chemotherapeutic agents through the phosphatase and tensin homolog (PTEN)–Akt–ABCG2 pathway. PTEN plays a crucial role in the self‐renewal and chemoresistance of liver T‐ICs; down‐regulation of PTEN by a lentiviral‐based approach reversed the effect of lupeol on liver T‐ICs. Using an in vivo chemoresistant HCC tumor model, lupeol dramatically decreased the tumor volumes of MHCC‐LM3 HCC cell line‐derived xenografts, and the effect was equivalent to that of combined cisplatin and doxorubicin treatment. Lupeol exerted a synergistic effect without any adverse effects on body weight when combined with chemotherapeutic drugs. Conclusion: Our results suggest that lupeol may be an effective dietary phytochemical that targets liver T‐ICs. (HEPATOLOGY 2011.)

Caveolin-1 overexpression is associated with hepatocellular carcinoma tumourigenesis and metastasis†

Caveolin‐1 (Cav1) has been implicated in diverse human cancers, yet its role in hepatocellular carcinoma (HCC) tumourigenesis and metastasis remains elusive. In the current study, we aim to provide a comprehensive understanding regarding the functional role of Cav1 in HCC tumourigenesis and metastasis. Cav1 expression was examined in a panel of human HCC cell lines using western blotting analysis and quantitative RT‐PCR and human tissues by immunohistochemistry. Cav1 was not detected in normal liver cell line and all non‐tumourous liver tissues but exclusively expressed in HCC cell lines and tissues. Dramatic expression of Cav1 was found in metastatic HCC cell lines and tumours, indicating a progressive increase of Cav1 expression along disease progression. Cav1 overexpression was significantly correlated with venous invasion (p = 0.036). To investigate the functions of Cav1 in HCC, Cav1 overexpressing and knockdown stable clones were established in HCC cells and their tumourigenicity and metastatic potential were examined. Overexpression of Cav1 promoted HCC cell growth, motility, and invasiveness, as well as tumourigenicity in vivo. Conversely, knockdown of Cav1 in metastatic HCC cells inhibited the motility and invasiveness and markedly suppressed the tumour growth and metastatic potential in vivo. Collectively, our findings have shown the exclusive expression of Cav1 in HCC cell lines and clinical samples and revealed an up‐regulation of Cav1 along HCC progression. The definitive role of Cav1 in promoting HCC tumourigenesis was demonstrated, and we have shown for the first time in a mouse model that Cav1 promotes HCC metastasis. Copyright

Liver tumor-initiating cells as a therapeutic target for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a common malignancy worldwide and has poor prognosis. Existing treatment modalities, including surgery, chemotherapy, and radiofrequency ablation, which target tumor bulk, have demonstrated limited therapeutic efficacy. In the past 10years, accumulating evidence has supported the existence of cancer stem cells (CSCs) or tumor initiating cells (T-ICs) within tumors including HCC. Identification of liver T-ICs and the signaling pathways that they are involved in may shed light on novel therapeutic strategies against this deadly disease. In this review, we will discuss recent progresses made in the research of liver T-ICs with regard to identification, functional characterization, regulation and therapeutic implications.

Nuclear factor kappa B–mediated CD47 up-regulation promotes sorafenib resistance and its blockade synergizes the effect of sorafenib in hepatocellular carcinoma in mice

Sorafenib is a new standard treatment for patients with advanced hepatocellular carcinoma (HCC). However, the survival benefit of this treatment is modest, partly owing to drug resistance. Recent evidence has demonstrated the existence of tumor‐initiating cells (T‐ICs) as the culprit for treatment resistance. To examine whether sorafenib resistance was a result of the presence of liver T‐ICs, we developed sorafenib‐resistant HCC cells both in vitro and in vivo through continuous exposure to sorafenib. Using these models, we found that sorafenib‐resistant clones demonstrated enhanced T‐IC properties, including tumorigenicity, self‐renewal, and invasiveness. In addition, several T‐IC markers were found to be up‐regulated, among which CD47 was found to be most significant. Using chromatin immunoprecipitation assays and expression analyses, CD47 expression was found to be regulated by nuclear factor kappa B (NF‐κB) through a specific response element in the promoter of CD47, and the site occupancy and expression were increased and decreased upon stimulation and inhibition of NF‐κB, respectively. Consistently, NF‐κB was activated in sorafenib‐resistant HCC cells, and this finding was confirmed in clinical HCC samples, which showed a positive correlation between NF‐κB and CD47 expression. Functional characterization of CD47 in sorafenib‐resistant HCC cells was evaluated using a lentivirus‐based knockdown approach and showed increased sensitization to sorafenib upon CD47 knockdown. Furthermore, blockade of CD47 using anti‐CD47 antibody (Ab) showed a similar effect. Using a patient‐derived HCC xenograft mouse model, we found that anti‐CD47 Ab (500 μg/mouse) in combination with sorafenib (100 mg/kg, orally) exerted synergistic effects on tumor suppression, as compared with sorafenib and anti‐CD47 Ab alone. Conclusions: NF‐κB‐mediated CD47 up‐regulation promotes sorafenib resistance, and targeting CD47 in combination with sorafenib is an attractive therapeutic regimen for the treatment of HCC patients. (Hepatology 2015;62:534–545

Chemopreventive Effect of PSP Through Targeting of Prostate Cancer Stem Cell-Like Population

Recent evidence suggested that prostate cancer stem/progenitor cells (CSC) are responsible for cancer initiation as well as disease progression. Unfortunately, conventional therapies are only effective in targeting the more differentiated cancer cells and spare the CSCs. Here, we report that PSP, an active component extracted from the mushroom Turkey tail (also known as Coriolus versicolor), is effective in targeting prostate CSCs. We found that treatment of the prostate cancer cell line PC-3 with PSP led to the down-regulation of CSC markers (CD133 and CD44) in a time and dose-dependent manner. Meanwhile, PSP treatment not only suppressed the ability of PC-3 cells to form prostaspheres under non-adherent culture conditions, but also inhibited their tumorigenicity in vivo, further proving that PSP can suppress prostate CSC properties. To investigate if the anti-CSC effect of PSP may lead to prostate cancer chemoprevention, transgenic mice (TgMAP) that spontaneously develop prostate tumors were orally fed with PSP for 20 weeks. Whereas 100% of the mice that fed with water only developed prostate tumors at the end of experiment, no tumors could be found in any of the mice fed with PSP, suggesting that PSP treatment can completely inhibit prostate tumor formation. Our results not only demonstrated the intriguing anti-CSC effect of PSP, but also revealed, for the first time, the surprising chemopreventive property of oral PSP consumption against prostate cancer.

Gene delivery using a receptor-mediated gene transfer system targeted to hepatocellular carcinoma cells

For gene therapy to be effective in cancers, it is necessary to deliver therapeutic genes into cells with high specificity and efficiency. In this study, we examined the in vitro and in vivo gene delivery efficiency of a new, growth receptor‐mediated gene transfer system in hepatocellular carcinoma (HCC). The effects of transfection of wild‐type p53 using this system were also studied. The system consisted of a ligand oligopeptide for epidermal growth factor receptor (EGFR) recognition, a polypeptide for DNA binding, and an endosome‐releasing oligopeptide for endosomolysis. Two human HCC cell lines and a normal liver cell line were used, and pCMV‐β‐galactosidase (β‐gal) was used as a reporter gene. Both HCC cell lines had strong expression of EGFR and the in vitro transfer efficiency peaked at day 5 at about 50%. This finding was in contrast to the normal liver cell line, which had weak EGFR expression and less than 1% transfer efficiency throughout. For in vivo gene transfer in tumors produced by inoculating HCC cells in nude mice and with the vector‐β‐gal gene complex injected peritumorally, β‐gal expression was detected within the tumors at 12 hr, peaked at day 5 involving about 50% of the tumor cells and persisted at 2 weeks. Using this vector system, transfection of wild‐type p53 into Huh‐7 cells that had mutated p53 resulted in significant growth inhibition of cancer cells accompanied by a decreased G2/M phase and increased p53 protein. In conclusion, this receptor‐mediated gene transfer system appears to work specifically in HCC cells with high efficiency, and may be promising in delivering apoptotic and other genes into HCC cells.