Anti-tumor activity and mechanism of oligoclonal hepatocellular carcinoma tumor-infiltrating lymphocytes in vivo and in vitro.

Objective: To explore a method for culturing hepatocellular carcinoma and tumor-infiltrating lymphocytes (HCC-TIL) and investigate the mechanism of TIL in killing tumors. Methods: The distribution of regulatory T cells (Treg) in HCC was detected by immunohistochemistry. Conventional TIL and oligoclonal TIL were isolated by the traditional method of enzyme digestion combined with mechanical treatment for whole HCC and micro HCC tissue block culturing method. MTT was used to compare the killing activity of TIL. Flow cytometry was used to analyze the proportion of CD8+ T cells and Treg cells in TIL. Tumor-bearing mice were established, and TIL adoptive immunotherapy was performed. Results: Treg cells were mainly distributed in the stroma of HCC. In vitro experiments showed oligoclonal TIL had higher cytotoxicity to tumor cells which negatively correlated with the proportion of Treg cells. In vivo experiments showed oligoclonal TIL had a higher anti-tumor effect. IFN-γ in peripheral blood and the positive rate of intratumoral lymphocytic infiltration in oligoclonal TIL group were both higher. TGF-ß and IL-10 in peripheral blood and the positive rate of intratumoral FoxP3 and IL-17 were both lower than those in conventional TIL group. Conclusion: The oligoclonal TIL culture method could obtain TIL with higher purity, and cytotoxicity to tumor cells was associated with Treg cells. The oligoclonal TIL had cytotoxicity to autologous HCC cells and significant inhibitory effect on the growth of transplanted tumors. The mechanism might be associated with the inhibition of Treg cells proliferation, increase of IFN-γ secretion, and decrease of TGF-ß, IL-10, and IL-17 secretion.

Improved method increases sensitivity for circulating hepatocellular carcinoma cells.

AIM: To improve an asialoglycoprotein receptor (ASGPR)-based enrichment method for detection of circulating tumor cells (CTCs) of hepatocellular carcinoma (HCC). METHODS: Peripheral blood samples were collected from healthy subjects, patients with HCC or various other cancers, and patients with hepatic lesions or hepatitis. CTCs were enriched from whole blood by extracting CD45-expressing leukocytes with monoclonal antibody coated-beads following density gradient centrifugation. The remaining cells were cytocentrifuged on polylysine-coated slides. Isolated cells were treated by triple immunofluorescence staining with CD45 antibody and a combination of antibodies against ASGPR and carbamoyl phosphate synthetase 1 (CPS1), used as liver-specific markers, and costained with DAPI. The cell slide was imaged and stained tumor cells that met preset criteria were counted. Recovery, sensitivity and specificity of the detection methods were determined and compared by spiking experiments with various types of cultured human tumor cell lines. Expression of ASGPR and CPS1 in cultured tumor cells and tumor tissue specimens was analyzed by flow cytometry and triple immunofluorescence staining, respectively. RESULTS: CD45 depletion of leukocytes resulted in a significantly greater recovery of multiple amounts of spiked HCC cells than the ASGPR(+) selection (Ps < 0.05). The expression rates of either ASGPR or CPS1 were different in various liver cancer cell lines, ranging between 18% and 99% for ASGPR and between 9% and 98% for CPS1. In both human HCC tissues and liver cancer cell lines, there were a few HCC cells that did not stain positive for ASGPR or CPS1. The mixture of monoclonal antibodies against ASGPR and CPS1 identified more HCC cells than either antibody alone. However, these antibodies did not detect any tumor cells in blood samples spiked with the human breast cancer cell line MCF-7 and the human renal cancer cell line A498. ASGPR(+) or/and CPS1(+) CTCs were detected in 29/32 (91%) patients with HCC, but not in patients with any other kind of cancer or any of the other test subjects. Furthermore, the improved method detected a higher CTC count in all patients examined than did the previous method (P = 0.001), and consistently achieved 12%-21% higher sensitivity of CTC detection in all seven HCC patients with more than 40 CTCs. CONCLUSION: Negative depletion enrichment combined with identification using a mixture of antibodies against ASGPR and CPS1 improves sensitivity and specificity for detecting circulating HCC cells.

CXCL8, overexpressed in colorectal cancer, enhances the resistance of colorectal cancer cells to anoikis.

Anoikis is a form of apoptosis which occurs when anchorage-dependent cells either show loss of adhesion or inappropriate adhesion. Only a few cancer cells that detach from the primary site of the tumor acquire the ability to resist anoikis and form metastasis. The mechanism underlying the resistance of colorectal cancer (CRC) cells to anoikis remains unclear. Interleukin-8 (alternatively known as CXCL8) is associated with CRC angiogenesis and progression. Here, we found that a high abundance of CXCL8 or TOPK strongly correlated with poor overall and disease-free survival of 186 patients with CRC. A combination of high CXCL8 and high TOPK expressions had the worst prognosis. We showed that CXCL8 expression was negatively correlated with anoikis in CRC cells. CXCL8 treatment enhanced the resistance of CRC cells to apoptosis, which was accompanied by the increase of TOPK, and the activation of AKT and ERK. Moreover, we demonstrated that the inhibition of either ERK or AKT by specific chemical inhibitors attenuated the CXCL8-mediated resistance to anoikis. Treatment with AKT inhibitor abolished the effects of CXCL8 on TOPK expression, suggesting that TOPK was downstream of AKT in the process of anoikis. Taken together, we demonstrated that CXCL8 is strongly implicated in the resistance of CRC cells to anoikis, and that the AKT, TOPK and ERK pathway may be a potential therapeutic target for CRC.

CCL20 and CXCL8 synergize to promote progression and poor survival outcome in patients with colorectal cancer by collaborative induction of the epithelial-mesenchymal transition.

Liver metastases represent the major cause of death in patients with colorectal cancer (CRC). Recent studies have suggested that the chemotactic responses of tumor cells are necessary for metastatic spread to the liver, and CCL20 and CXCL8 have a strong association with CRC metastasis. The aim of our study was to identify the mechanisms by which CCL20 and CXCL8 synergize to promote metastatic progression and evaluated their potential as prognostic markers for CRC patients. The abilities of CCL20 and CXCL8 to promote CRC cell progression and epithelial-mesenchymal transition(EMT)phenotype were analyzed in vitro. Possible signaling pathways were investigated with specific pathway inhibitors and small interfering RNA (siRNA). 213 Patients with CRC who underwent surgery were enrolled for analysis of CCL20, CXCL8 and E-cadherin expressions in tumor tissues. Prognostic factors were then identified. CCL20 or CXCL8 alone was not sufficient to induce complete EMT in CRC cells, but both of them could coordinately induce EMT-like phenotype that was required to maintain CRC cell proliferation, migration and invasion. PI3K/AKT-ERK1/2 pathway crosstalk was demonstrated to be responsible for this process. Coexpression of CCL20 and CXCL8 was negatively correlated with E-cadherin expression in human CRC tissues. CRC patients with coexpression of CCL20 and CXCL8 were more likely to develop liver metastases and both coexpression was an independent high-risk factor for a most poor prognosis. CCL20 and CXCL8 synergize to promote CRC metastatic progression by coordinated induction of EMT via PI3K/AKT-ERK1/2 signaling axis. Detection of both coexpressions can be used to predict clinical outcomes in CRC patients.

Targeting of circulating hepatocellular carcinoma cells to prevent postoperative recurrence and metastasis.

Currently, the main treatment for hepatocellular carcinoma (HCC) involves the surgical removal of tumors or liver transplantation. However, these treatments are often not completely curative, as they are associated with a risk for postoperative recurrence and metastasis. Circulating tumor cells (CTCs) are increasingly recognized as the main source for recurrence and metastasis after radical hepatectomies are performed. Many studies have demonstrated the association between the presence of either pre- or postoperative CTCs and an increased risk for HCC recurrence. To improve the therapeutic outcome of HCC, a personalized, comprehensive and multidisciplinary approach should be considered, involving the application of appropriate diagnostic and therapeutic measures targeting HCC CTCs in different stages throughout the course of treatment. This article proposes some HCC CTC-based strategies for the treatment of HCC, including the monitoring of HCC CTCs before, during and after radical hepatectomy, therapeutic targeting of HCC CTCs, prevention of the generation and colonization of CTCs, as well as the use of CTC indexes for the selection of indications, prediction of prognoses, and planning of individualized therapeutic regimens. Innovation and technological development of therapies targeting CTCs, as well as their translation into clinical practice, will help to effectively reduce postoperative recurrence and metastasis, and significantly prolong the survival of HCC patients.

Changes of serum alpha-fetoprotein and alpha-fetoprotein-L3 after hepatectomy for hepatocellular carcinoma: prognostic significance.

BACKGROUND: Alpha-fetoprotein (AFP) is the most established tumor marker of hepatocellular carcinoma (HCC), but one of its limitations is non-specificity. Many studies have demonstrated that alpha-fetoprotein-L3 (AFP-L3) is more specific than AFP in the early diagnosis and prognosis of HCC. However, there is a lack of knowledge about the post-hepatectomy profiles of serum AFP and AFP-L3 values in HCC patients. To identify the profiles after surgical resection of HCC, we analyzed the correlation between the profiles and postoperative HCC recurrence or survival, and evaluated their utility in predicting postoperative therapeutic efficacy and prognosis. METHODS: From August 2003 to December 2004, 318 patients with positive serum AFP who had received surgical resections were enrolled in this study. Preoperative and postoperative serum AFP and AFP-L3 levels were measured simultaneously and regularly, and their postoperative profiles during a long-term follow-up were recorded and summarized. RESULTS: A high ratio of AFP-L3 to total AFP was shown to correlate with pathologic features of aggressiveness. The overall 1-, 3-, and 5-year recurrence rates of the whole series were 28%, 57%, and 84%, and the overall survival rates were 86%, 61%, and 33%, respectively. The changes of serum AFP and AFP-L3 after hepatectomy for HCC were classified into 3 groups (group A: AFP-L3 undetectable; group B: AFP-L3 <10%; and group C: AFP-L3 >10%). Patients with positive postoperative AFP-L3 had significantly earlier recurrence than those with negative results. The overall survival was significantly shorter in the positive groups than in the groups negative for postoperative AFP-L3. CONCLUSION: Post-hepatectomy changes in serum AFP and AFP-L3 levels occurred in three distinct patterns, which were closely correlated with HCC recurrence and patient survival with different prognostic values.

Expression of HBx protein in hepatitis B virus-infected intrahepatic cholangiocarcinoma.

BACKGROUND: Hepatitis B virus (HBV) is an etiological factor of intrahepatic cholangiocarcinoma (ICC), but the pathogenic mechanisms remain unclear. This study aimed to investigate the expression and possible role of HBx, an HBV-encoded potentially oncogenic protein, in HBV-infected ICC. METHODS: Tissue samples were obtained from 54 specimens of HBV-infected ICC. Forty-four specimens were of peripheral type and 10 hilar type. Formalin-fixed, paraffin-embedded sections of the specimens were immunohistochemically stained for HBx and p53. RESULTS: HBx expression was found in 70.4% (38/54) of the specimens, and it was more frequently seen in the peripheral type than in the hilar type (79.5% vs 30.0%, P=0.002). All three well-differentiated ICCs expressed HBx, whereas 76.9% (30/39) moderately-differentiated and 41.7% (5/12) poorly-differentiated ICCs had HBx expression (P=0.033). Patients with HBx expression had a significantly higher prevalence of elevated serum alpha-fetoprotein (P=0.033). p53 protein expression was found in 18 of 54 cases (33.3%), and was not correlated with that of HBx. CONCLUSIONS: HBx may contribute to the pathogenesis of ICC, particularly the peripheral type. p53 abnormality may not play a significant role in HBx-mediated oncogenicity during ICC carcinogenesis.

MCM7 expression predicts post-operative prognosis for hepatocellular carcinoma.

BACKGROUND: Dysregulation of minichromosome maintenance protein 7 (MCM7) was previously identified in multiple human malignancies. The clinical significance of MCM7 expression is yet to be delineated in patients with hepatocellular carcinoma (HCC). METHODS: Paired cancerous and non-cancerous specimens from 87 patients with HCC who underwent resection were used for the immunohistochemical evaluation of MCM7 expression. Effect of sorafenib on the expression of MCM7 was tested in two human HCC cell lines SMMC-7721 and PLC/PRF/5. RESULTS: Non-cancerous tissues were negative for immunohistochemical staining for MCM7 expression. Nuclear MCM7 was expressed in 42 of 87 HCC (48.2%) and was correlated with hepatitis B virus infection (P = 0.020), intrahepatic metastasis (P = 0.022) and vascular invasion (P = 0.013). Moreover, its expression was correlated with shorter overall survival (P = 0.033). Multivariate analysis showed that MCM7 expression was an independent prognostic factor for overall survival(P = 0.041). Sorafenib inhibited the expression of MCM7 in a concentration-dependent manner in vitro. CONCLUSIONS: The current findings suggested that MCM7 expression may be a useful predictor of prognosis in patients with HCC after resection. Adjuvant therapy with sorafenib might be a valuable therapeutic strategy for MCM7-positive HCC patients.

Clinicopathological significance of ZEB1 protein in patients with hepatocellular carcinoma.

BACKGROUND: ZEB1, a member of the ZFH family of proteins (zinc-finger E-box binding homeobox), plays a central role in epithelial-mesenchymal transition (EMT) during carcinogenesis. In this study, we investigated the expression of ZEB1 in patients with hepatocellular carcinoma (HCC) and its clinical effects with underlying mechanisms. METHODS: Expression levels of ZEB1 were assessed by Western blot in 5 HCC cell lines and in paired cancerous and noncancerous tissues from 110 patients with HCC. Short-hairpin RNA (shRNA) interference for ZEB1 was performed in MHCC-97H cell line. RESULTS: ZEB1 protein was detected at a relatively high level in metastatic human HCC cell lines (MHCC-97L and MHCC-97H) when compared with that in nonmetastatic HCC cell lines (Hep3B, PLC and Huh-7). ZEB1 was expressed at high levels in 72 of 110 HCC patients (65.4%) and correlated with advanced TNM stage, tumor size >5 cm, intrahepatic metastasis, vascular invasion, and frequent early recurrence. The results of multivariate analysis revealed that ZEB1 high expression was a significant prognostic factor for poor overall and disease-free survivals. Silencing ZEB1 resulted in significant suppression of motility of MHCC-97H cell line, which was accompanied with increased expression of the epithelial marker E-cadherin and decreased expression of the mesenchymal markers N-cadherin and vimentin. Furthermore, silencing ZEB1 prevented the spread of intrahepatic metastasis and increased overall survival in mouse orthotopic tumor models. CONCLUSIONS: This study shows that ZEB1 high expression was correlated with HCC malignant progression and subsequent poor patient survival by induction of EMT changes.

Risk factors of intrahepatic cholangiocarcinoma in patients with hepatolithiasis: a case-control study.

BACKGROUND: Why 3.3% to 10% of all patients with hepatolithiasis develop intrahepatic cholangiocarcinoma (ICC) remains unknown. We carried out a hospital-based case-control study to identify risk factors for the development of ICC in patients with hepatolithiasis in China. METHODS: Eighty-seven patients with pathologically diagnosed hepatolithiasis associated with ICC and 228 with hepatolithiasis alone matched by sex, age (+/-2 years), hospital admittance and place of residence were interviewed during the period of 2000-2008. Odds ratios (OR) and 95% confidence intervals (CI) were calculated for each risk factor. RESULTS: Among the patients with hepatolithiasis associated with ICC, the mean age was 57.7 years and 61.0% were female. Univariate analysis showed that the significant risk factors for ICC development in hepatolithiasis were smoking, family history of cancer, appendectomy during childhood (under age 20), and duration of symptoms >10 years. In multivariate stepwise logistic regression analysis, smoking (OR=1.931, 95% CI: 1.000-3.731), family history of cancer (OR=5.175, 95% CI: 1.216-22.022), and duration of symptoms >10 years (OR=2.348, 95% CI: 1.394-3.952) were independent factors. CONCLUSION: Smoking, family history of cancer and duration of symptoms >10 years may be risk factors for ICC in patients with hepatolithiasis.

Isolation of circulating tumor cells in patients with hepatocellular carcinoma using a novel cell separation strategy.

PURPOSE: To establish a sensitive and specific isolation and enumeration system for circulating tumor cells (CTC) in patients with hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: HCC cells were bound by biotinylated asialofetuin, a ligand of asialoglycoprotein receptor, and subsequently magnetically labeled by antibiotin antibody-coated magnetic beads, followed by magnetic separation. Isolated HCC cells were identified by immunofluorescence staining using Hep Par 1 antibody. The system was used to detect CTCs in 5 mL blood. Blood samples spiked with Hep3B cells (ranging from 10 to 810 cells) were used to determine recovery and sensitivity. Prevalence of CTCs was examined in samples from HCC patients, healthy volunteers, and patients with benign liver diseases or non-HCC cancers. CTC samples were also analyzed by FISH. RESULTS: The average recovery was 61% or more at each spiking level. No healthy, benign liver disease or non-HCC cancer subjects had CTCs detected. CTCs were identified in 69 of 85 (81%) HCC patients, with an average of 19 ± 24 CTCs per 5 mL. Both the positivity rate and the number of CTCs were significantly correlated with tumor size, portal vein tumor thrombus, differentiation status, and the disease extent as classified by the TNM (tumor-node-metastasis) classification and the Milan criteria. HER-2 gene amplification and TP53 gene deletion were detected in CTCs. CONCLUSION: Our system provides a new tool allowing for highly sensitive and specific detection and genetic analysis of CTCs in HCC patients. It is likely clinically useful in diagnosis and monitoring of HCC and may have a role in clinical decision making.

Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein as a marker of prognosis and a monitor of recurrence of hepatocellular carcinoma after curative liver resection.

BACKGROUND: The aim of this study was to determine the role of Lens culinaris agglutinin-reactive fraction of alpha-fetoprotein (AFP-L3) as a prognostic marker and a monitor marker of recurrence after curative resection of hepatocellular carcinoma (HCC). METHODS: From December 2002 to May 2004, 395 consecutive patients with HCC who underwent curative partial hepatectomy were included in the study. The tumor characteristics and clinical outcomes of patients with positive preoperative and postoperative AFP-L3 were compared with those with negative results. RESULTS: A high ratio of AFP-L3 to total AFP was an indicator of pathologic aggressiveness. Patients with positive preoperative AFP-L3 had significantly earlier recurrence (median time to recurrence 22.0 ± 2.4 months vs 45.0 ± 6.9 months, P < .001) when compared with those with negative preoperative results. Significantly more patients with continuously positive or negative-turn-positive AFP-L3 results after surgery developed recurrence, particularly distant metastases, when compared with patients with continuously negative AFP-L3 results. The overall and disease-free survivals were significantly shorter in the positive than the negative preoperative AFP-L3 group. The overall and disease-free survivals were significantly shorter in the continuously positive and the negative-turn-positive than the continuously negative postoperative AFP-L3 group. CONCLUSION: Positive preoperative AFP-L3 and continuously positive or negative-turn-positive AFP-L3 results after surgery predicted a more aggressive tumor behavior, higher tumor recurrence, and poorer clinical outcomes. HCC patients with an increased proportion of AFP-L3 to total AFP should be more aggressively treated and closely followed-up.

Risk factors for early recurrence of small hepatocellular carcinoma after curative resection.

BACKGROUND: Poorer prognosis is seen in patients with hepatocellular carcinoma (HCC) after curative hepatic resection with early recurrence (1 year). This study aimed to identify risk factors for postoperative early recurrence of small HCC (100 ng/ml, lack of tumor capsule formation, microscopic vascular invasion, high Edmonson-Steiner grades, and cytokeratin-19 (CK-19) expression (P<0.05). Multivariate stepwise logistic regression analysis showed that serum AFP level >100 ng/ml (odds ratio 2.561, 95% confidence interval 1.057 to 6.206, P=0.037) and microscopic vascular invasion (odds ratio 4.549, 95% confidence interval 1.865 to 11.097, P=0.001) were independent factors. CONCLUSIONS: Postoperative early recurrence is related to serum AFP level >100 ng/ml and microscopic vascular invasion in patients with small HCC. Adjuvant therapy and careful follow-up are required for patients with these risk factors.

[Progress and prospects in cancer stem cell research for hepatocellular carcinoma].

The cancer stem cell (CSC) theory stipulates that it is a small population of cells called CSCs that initiates tumor formation and maintains its growth. CSCs are scarce within the bulk of the tumor mass, and possess stem cell-like properties such as self-renewal, differentiation and resistance to therapies, and so on. In the past few years, by using side population technique and approaches based on surface markers, including CD133, CD90, OV6 and EpCAM, researchers have identified and isolated a subpopulation of liver cancer cells with enhanced colony-forming and tumorigenic ability, which is strong evidence for the existence of liver CSCs. In this review, we summarized the progress of research on liver CSCs, discussed the significance of liver CSCs in the diagnosis and treatment of hepatocellular carcinomas, and put forward the future research directions as well as the challenges and opportunities.

Troglitazone, a peroxisome proliferator-activated receptor gamma ligand, induces growth inhibition and apoptosis of HepG2 human liver cancer cells.

AIM: To examine the effect of troglitazone, a peroxisome proliferator-activated receptor gamma (PPAR gamma) ligand, on the proliferation and apoptosis of human liver cancer cells. METHODS: Liver cancer cell line HepG2 was cultured and treated with troglitazone. Cell proliferation was detected by 3-(4-,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay; apoptosis was detected by flow cytometry and terminal deoxynucleotidyl transferase-mediated nick end labeling of DNA fragmentation sites (TUNEL) assay; and apoptosis-related protein was detected by immunocytochemistry and Western blotting. RESULTS: Troglitazone inhibited growth and induced apoptosis of HepG2 cells in a dose-dependent manner, and induced activation of caspase-3 expression. Troglitazone not only drove apoptosis-inhibiting factor survivin to translocate incompletely from the nucleus to the cytoplasm, but also inhibited expression of survivin, while it did not affect expression of apoptosis-promoting factor Bax. CONCLUSION: PPAR gamma ligands inhibit growth and induce apoptosis of liver cancer cells, and may have applications for the prevention and treatment of liver cancer.

Clinicopathologic characteristics of intrahepatic cholangiocarcinoma in patients with positive serum a-fetoprotein.

AIM: To explore clinicopathologic characteristics of intrahepatic cholangiocarcinoma (ICC) in patients with positive serum a-fetoprotein (AFP). METHODS: One hundred and thirty one patients who underwent surgical dissection for pathologically confirmed ICC were divided into a positive AFP (> 20 ng/mL) group (n = 32) and a negative AFP group (n = 99), whose clinicopathologic features were analyzed and compared. RESULTS: The positive rate of HBsAg and liver cirrhosis of the positive AFP group was higher than that of the negative AFP group, while the positive rate of CA19-9 (> 37 U/mL) and the lymph node metastasis rate was lower. CONCLUSION: ICC patients with positive AFP share many clinicopathologic similarities with hepatocellular carcinoma.

Risk factors for intrahepatic cholangiocarcinoma: a case-control study in China.

AIM: To carry out a hospital-based case-control study to investigate risk factors for intrahepatic cholangiocarcinoma (ICC) in China. METHODS: A total of 312 ICC cases and 438 matched controls were included in the study. The presence of diabetes mellitus, hypertension, hepatolithiasis, primary sclerosing cholangitis, liver fluke infection (Clonorchis sinensis), was investigated through clinical records. Blood from all participants was tested for hepatitis B surface antigen (HBsAg) and anti-HCV antibodies. Odds ratios (OR) and 95% confidence intervals (95% CI) were estimated using conditional logistic regression. RESULTS: Compared with controls, ICC patients had a higher prevalence of HBsAg seropositivity (48.4% vs 9.6%, P<0.000), and hepatolithiasis (5.4% vs 1.1%, P=0.001). By multivariate analysis, the significant risk factors for development of ICC were HBsAg seropositivity (adjusted OR, 8.876, 95% CI, 5.973-13.192), and hepatolithiasis (adjusted OR, 5.765, 95% CI, 1.972-16.851). The prevalence of anti-HCV seropositivity, diabetes mellitus, hypertension, cigarette smoking, and alcohol consumption were not significantly different between cases and controls. CONCLUSION: These findings suggest that HBV infection and hepatolithiasis are strong risk factors for development of ICC in China.

[Application of the improved MTT assay in predicting the intrinsic drug resistance of liver cancer].

OBJECTIVE: To investigate the role of the improved MTT assay in prediction of intrinsic drug resistance of liver cancer. METHODS: The convenient MTT colorimetry was innovated to test the effects of 4'-epi-adriamycin (E-ADM), carboplatin (CBP), and 5-Fluorouracil(5-Fu), used alone or in combination, on 30 specimens of primary liver cancer without chemotherapy. All of the 30 paraffin-embedded tissues were assembled in a microarray. The used terminal concentrations of drugs were one twentieth those of the plasma peak concentrations calculated by using the liver cancer cells of the line SMMC-7721. The expression of P-glycoprotein (P-gp), multidrug resistant protein (MRP)-3, lung resistance-related protein (LRP), glutathione S-transferase (GST)-pi, and 2 kinds of cyclin-related protein: p16(INK4a) and p21WAF1, were detected by immunohistochemistry. RESULTS: Sixteen of the 30 specimens (53.3%) were drug-resistant and 14 of the 30 specimens (46.7%) were drug-sensitive. The sequence of drug -sensitivity was in the order of combination chemotherapy, E-ADM, 5-Fu, and CBP. The positive rate of P-gp in the drug-resistant group was 56.3%, significantly higher than that of the drug-sensitive group (14.3%, P < 0.05). CONCLUSION: An improved MTT assay has been developed that is more scientific and worth spreading clinically. Intrinsic drug resistance of liver cancer is popular. P-gp is a good predictive marker in intrinsic drug resistance of liver cancer.