| Hepatocellular carcinoma (HCC) is the fifth most frequent malignant tumors, and the third leading cause of cancer-related mortality in the world. The desease incidence and mortality of HCC in China are higher than other country. HCC patients are usually diagnosed when the tumor is in an advanced stage and lose the opportunity for curative surgery. Other treatments including loco-regional or systemic chemotherapy, fail mainly due to the chemoresistance of tumor and inability to endure treatment responses.One of the most commonly used chemotherapy drugs for HCC is doxorubicin (DOX), but high doses of DOX result in severe toxicities, such as hematological, gastro-intestinal, renal, hepatic toxicities, and particularly cardiac toxicities.Increasing evidence supports the role of cathepsin B (Cat B) in tumor invasion and metastasis, including HCC progression. Cat B expression is increased in many cancers at the mRNA, protein and activity levels, and closely related to invasive behavior of cancer. Therefore, Cat B could be a potential target for new drugs designed specially against invading cancer cells.To retain the therapeutic effect while reducing the toxicity of DOX, Dubowchik et al. designed a smart prodrug of DOX, Ac-Phe-Lys-PABC-DOX (PDOX), in which a Cat B-specific dipeptide is introduced, along with a spacer PABC (para-aminobenzyloxycarbonyl) to increase the distance between dipeptide and DOX, so that the dipeptide can enter the Cat B’ active site. As a result of this molecular re-structuring, the prodrug is inactive in blood circulation and normal tissues where little Cat B exists in the active form. When the prodrug reaches Cat B-enriched area such as the invasion front of cancer, the Phe-Lys dipeptide is cleaved by Cat B, exposing the PABC spacer that is then hydrolyzed spontaneously, releasing free DOX at the cancer invasion front. Thus this prodrug could exert cytotoxicity to invading cancer cells while protecting normal cells from excessive drug exposure, a strategy called passive targeted therapy.In our previous animal model study, we investigated the activities and side effect of PDOX to treat peritoneal carcinomatosis (PC) from gastric cancer, which suggests that PDOX might be a promising new drug against cancer invasion. Inspired by the initial results, we designed this study to further explore the treatment potential of this prodrug in a mord aggressive and highly lethal orthotopic nude mice model of HCC.Part I.To investigate the efficacy of PDOX to liver-orthotopic nude mice model with highly metastatic human hepatocellular carcinoma.Objective To investigate the efficacy of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug. Methods Highly metastatic human HCC cell line HCCLM9was used for animal model construction. Briefly, cells (5X106cells each) in0.1ml phosphate buffered saline (PBS) were injected subcutaneously into the left flank of2nude mice after HCCLM9cells were successly cultivated. The subcutaneous tumors were removed when they reached lcm or so in diameter, and minced into pieces (1mm3) to perform orthotopic transplantation into livers of male nude mice (n=33). On day8after the model establishment, the animals were randomized into Control group (n=10), DOX group (n=11), and PDOX group (n=12), and treated with PDOX, DOX, and saline through tail vein, respectively. The animals were weighed every four days, and were treated every eight days. At the endpoint of experiment, two nude mice in the Control group died. All animals were subjected to magnetic resonance image (MRI) study to measure the liver tumor size. Then the mice were anesthetized by peritoneal injection of3%phenobarbital chloride. The peripheral blood was obtained for alpha fetoprotein (AFP) test. Tumor tissues and major organs including the heart, the liver, the lungs and any other suspected organs were collected, fixed with4%paraformaldehyde and embedded in paraffin for pathological studies. At autopsy, liver tumor weight and size, ascites, abdominal lymph nodes metastases, experimental peritoneal carcinomatosis index (ePCI), and tumor-host body weight ratio were investigated. Results After tumor inoculation into the liver, the animals in the DOX and PDOX groups showed slight and progressive body weight decreases till the study endpoint. The general status of animals appeared better in the PDOX group than the DOX group, which in turn was better than Control group. In the Control group, the animals showed body weight increases from d30to d36, mainly due to excessive liver tumor and ascites. Prominent liver tumors were observed in all animals, and representative MRI abdominal scan of liver tumors were shown. Compared with Control, PDOX and DOX treatment reduced tumor weights by43.6%and42.0%, respectively. Similarly, PDOX and DOX treatments reduced tumor volumes by53.4%and49.1%,respectively (P<0.01, DOX/PDOX vs Control). The tumor-weight to body-weight ratio was also significantly reduced from27.94%in the Control group to18.28%in the DOX group and18.10%in the PDOX group (P<0.001, DOX/PDOX vs Control). The serum AFP level was reduced from97.27±34.22ng/ml in the Control group to24.69±12.09ng/ml in the DOX group and22.31±13.42ng/ml in the PDOX group (P<0.001, DOX/PDOX vs Control). The ePCI was reduced from9±2in the Control group to6±2in the DOX group and6±2in the PDOX group (P<0.05, DOX/PDOX vs Control). Another significant effect was observed on retroperitoneal lymphnode metastases, which occurred in80%(8/10),27.3%(3/11) and16.7%(2/12) of animals, respectively, in the Control, DOX, and PDOX groups (P <0.05, DOX/PDOX vs Control). The rates of animals with lung metastases were reduced from100.0%(10/10) in the Control group to63.6%(7/11) in the DOX group and33.3%(4/12) in the PDOX group (P<0.05, Control vs DOX; P<0.05, Control vs PDOX; P<0.05, DOX vs PDOX). Conclusion Compared with DOX, PDOX may have similar efficacy to primary tumor and loco-regional metastases, but have better anti-metastatic efficacy such as to lung metastasis. Part Ⅱ.To investigate the side effect of PDOX to liver-orthotopic nude model with highly metastatic human hepatocellular carcinoma.Objective To investigate the side effect of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug. Methods On d36, animals were euthanized, and blood was obtained for routine studies, including peripheral blood profiles by Sysmex KX-21automated hematology analyzer; liver function parameters alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl transpeptidase (GGT), total bilirubin levels (TBIL), and direct bilirubin (DBIL) levels; renal function parameters blood urea nitrogen (BUN) and creatinine (Cr) levels; cardiac function parameters creatine kinase (CK), creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH) levels; electrolytes (K+, Na2+Ca2+, Mg2+, and Cl-); all by Aeroset Clinical Chemistry Analyzer. Main organs obtained from animals of3groups were subjected to histopathological examination to detect the toxicity of drug. Results In peripheral blood routine, the white blood cells levels in PDOX mice were higher than DOX mice (1.98-folds, P<0.05). The platelet levels were higher in the PDOX group (1.67-folds, P<0.05) and the DOX group (1.59-folds, P<0.05) compared with Control. There were no differences in red blood cells and hemoglobin levels among the3groups. In terms of liver function, compare with Control, DOX and PDOX caused significant reduction in GGT and AST levels (P<0.05, DOX/PDOX vs Control). There were no statistically significant differences in ALT, TBIL and DBIL levels among the3groups. In terms of renal functions, compared with Control, both DOX and PDOX resulted in significant reduction in serum BUN levels (P<0.001, Control vs DOX; P<0.05, Control vs PDOX), and BUN levels in the PDOX group were also significantly lower than those in the DOX group (P<0.05). Furthermore, the serum Cr levels in the PDOX group were much lower than those of the Control and DOX groups (P<0.05, PDOX vs Control; P<0.05, PDOX vs DOX). Electrolytes results demonstrated that C1-was reduced in PDOX compared with Control group (P<0.05); but Ca2+was increased in PDOX compared with the Control and DOX groups (P<0.05, PDOX vs Control; P<0.05, PDOX vs DOX). Cardiac functions demonstrated that both DOX and PDOX significantly decreased LDH compared with Control group (P<0.05, DOX/PDOX vs Control), but there were no differences between the DOX and PDOX groups. Compared with Control, DOX increased CK and CK-MB levels, although the differences didn’t reach the statistical significance. On the other hand, PDOX significantly decreased CK, compared with DOX (P<0.05). Histopathological study revealed multiple spotty degenerative changes in the myocardium in DOX-treated mice. There were no observable histopathological changes in both Control and PDOX groups. Conclusion PDOX has less hematological and biochemical toxicities, especially less cardio-toxicity than DOX.Part Ⅲ. To investigate efficacious mechanism of PDOX to liver-orthotopic nude model with highly metastatic human hepatocellular carcinoma.Objective To investigate efficacious mechanism of cathepsin B-cleavable doxorubicin (DOX)-prodrug (PDOX) for targeting therapy of metastatic human hepatocellular carcinoma (HCC) using DOX as a positive control drug. Methods Tumor tissues obtained from animals of3groups were subjected to immunohistochemistry to detect the expressions of Cat B, Ki-67, CD34, VEGF, E-cadherin and D2-40. Fresh tumor tissues in RIPA lysis buffer were manually homogenized on ice, then centrifuged to remove cellular and nuclear debris. The protein concentration was determined using a BCA Assay kit. The concentrated proteins were determined the expressions of p-ERK1/2, ERK1/2, BCL-2, caspase-3, and β-actin using western blotting. Results For the evaluation of IHC results, positive cells were stained brownish granules in the cell membrane, cytoplasm or nucleus. In all cases, cytoplasmic Cat B expression was scaled as moderate and strong expression. Ki-67expressed in the nucleus. VEGF positive cells were stained both in the nucleus and cytoplasm. The expression of E-cadherin mainly existed in cell membrane and cytoplasm. CD34and D2-40positive cells were stained in cytoplasm. The expression of Ki-67was evaluated according to positive rate. The positive expression of CD34and D2-40was evaluated according to microvessel density (MVD) and lymphatic microvessel density (LMVD). Positive cytoplasmic Cat B expression was observed in all tumors from the3group. Ki-67positive rates were77.1±7.8%in the Control group,72.3±4.9%in the DOX group, and61.6±14.6%in the PDOX group (P<0.05, Control vs DOX; P<0.05, Control vs PDOX; P <0.05, DOX vs PDOX). The median MVD values of CD34were47.2(21.4-70.0) in the Control group,60.9(37.0-91.2) in the DOX group, and55.6(22.2-80.2) in the PDOX group, respectively (P>0.05). The VEGF positive rate was not statistically different among the3groups (P>0.05). Similarly, there was no statistical difference in the expression of E-cadherin among the3groups (P>0.05). The median values of LMVD designated as D2-40positive expression were0.5(0.0-3.2),1.8(0.0-8.4) and1.8(0.0-5.8) in the Control, DOX and PDOX groups, respectively (P>0.05). Western blotting showed that compared with Control, PDOX and DOX reduced ERK phosphorylation, decreased BCL-2expression, and increased caspase-3and caspase-9activation. Conclusion PDOX has better tumor inhibition than DOX. And PDOX produced the effect at least via ERK pathway. |
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