pyrophosphate and Carcinoma--Hepatocellular

We previously showed that human hepatic intrasinusoidal (HI) natural killer (NK) T cells selectively eliminate hepatocellular carcinoma (HCC) cell lines. In this study, we investigated the underlying mechanisms on how HI γδ T cells, expanded with zoledronate, exhibit a superior cytotoxic effect on HI NK-resistant Huh7 HCC cells.. γδ T cells were obtained from living liver transplant donors or from peripheral blood mononuclear cells (PBMC) of healthy volunteers and were expanded in the presence of IL-2, IL-15, and zoledronate for 2 weeks. Cytotoxicity was measured using the lactate dehydrogenase (LDH) assay in vitro and by flow cytometry using carboxyfluorescein succinimidyl ester (CFSE) in vivo.. The cytotoxicity of expanded HI γδ T cells against Huh7 cells was associated with a higher pyrophosphate expression in Huh7 cells compared to SNU398 cells. In contrast, the cytotoxicity of HI γδ T cells against SNU398 cells depended on NKG2D. HI γδ T cells expressed less PD-1 than PB γδ T cells. The cytotoxicity of HI γδ T cells against Du145 and PC3 prostate cancer cells was also associated with pyrophosphate expression in these cells, as well as NKG2D and DNAM-1.. The expression levels of phospho-antigen in tumor cells determined the cytotoxicity of HI γδ T cells, although the NK activating receptors, death ligands, and immune checkpoint molecules also contribute to their cytotoxicity. γδ T cells are attractive candidates for cancer immune cell therapy.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cytotoxicity, Immunologic; Diphosphates; Humans; Leukocytes, Mononuclear; Liver Neoplasms; Male; NK Cell Lectin-Like Receptor Subfamily K; Receptors, Antigen, T-Cell, gamma-delta; Zoledronic Acid

Cyclin-dependent kinase 9 (CDK9) inhibitors are a novel category of anticancer treatment for cancers. However, their effects on hepatocellular carcinoma (HCC) are rarely investigated. Human ribonucleotide reductase (RR, which consists of RRM1 and RRM2 subunits) catalyzes the conversion of ribonucleoside diphosphate into 2'-deoxyribonucleoside diphosphate to maintain the homeostasis of nucleotide pools, which play essential roles in DNA synthesis and DNA repair. In this study, we identified that CDK9 protein expression in adjacent non-tumor tissues predicted HCC patients' overall and progression-free survivals. The anticancer activity of a CDK9-selective inhibitor, LDC000067, on HCC cells was positively associated with its ability to inhibit the expression of RRM1 and RRM2. LDC000067 downregulated RRM1 and RRM2 expression through post-transcriptional pathway. Specifically, LDC000067 triggered RRM2 protein degradation via multiple pathways, including proteasome-, lysosome-, and calcium-dependent pathways. Furthermore, CDK9 positively correlates with RRM1 or RRM2 expression in HCC patients, and the expressions of these three genes were associated with the higher infiltration of immune cells in HCC. Taken together, this study identified the prognostic relevance of CDK9 in HCC and the molecular mechanism for the anticancer effect of CDK9 inhibitors on HCC.

Topics: Carcinoma, Hepatocellular; Cell Line, Tumor; Cyclin-Dependent Kinase 9; Diphosphates; Humans; Liver Neoplasms; Ribonucleotide Reductases

Topics: alpha-Fetoproteins; Carcinoma, Hepatocellular; Diphosphates; Humans; Liver Neoplasms; Mutation Rate; Recurrence; Retrospective Studies; Tumor Suppressor Protein p53

Hepatocellular carcinoma (HCC) and colorectal carcinoma with hepatic metastases (mCRC) are cancers with poor prognosis and limited therapeutic options. New approaches are needed and adoptive immunotherapy with Vgamma9Vdelta2 T lymphocytes represents an attractive strategy. Indeed, Vgamma9Vdelta2 T cells were shown to exhibit efficient lytic activity against various human tumor cell lines, and in vitro Vgamma9Vdelta2 T expansion protocol based on single phosphoantigen stimulation could be easily performed for healthy donors. However, a low proliferative response of Vgamma9Vdelta2 T cells was observed in about half of the cancer patients, leading to an important limitation in the development of Vgamma9Vdelta2 T cell-based immunotherapy. Here, for the first time in the context of cancer patients, Vgamma9Vdelta2 T cell expansions were performed by co-culturing peripheral blood mononuclear cell (PBMCs) with autologous dendritic cells (DCs) pretreated with aminobisphosphonate zoledronate. For patients not responding to the conventional culture protocol, co-culture of PBMC with zoledronate-pretreated DCs induced strong cell expansion and allowed reaching a minimal rate of purity of 70% of Vgamma9Vdelta2 T cells. The potent immunostimulatory activity of zoledronate-treated DCs was associated with higher amount of isopentenyl pyrophosphate (IPP) in the culture and was correlated with better ability to activate Vgamma9Vdelta2 T cells as measured by IFN-gamma production. Moreover, we demonstrated that the cytotoxic level of Vgamma9Vdelta2 T cells against freshly autologous tumor cells isolated from patients could be significantly increased by pretreating the tumor cells with zoledronate. Thus, this method of generating Vgamma9Vdelta2 T cells leads eligible for Vgamma9Vdelta2 T cell adoptive immunotherapy the HCC and mCRC patients.

Topics: Adenocarcinoma; Aged; Blotting, Western; Bone Density Conservation Agents; Carcinoma, Hepatocellular; Cell Differentiation; Cell Proliferation; Coculture Techniques; Colorectal Neoplasms; Cytotoxicity, Immunologic; Dendritic Cells; Diphosphates; Diphosphonates; Female; Flow Cytometry; Hemiterpenes; Humans; Imidazoles; Immunotherapy, Adoptive; Liver Neoplasms; Male; Middle Aged; Organophosphorus Compounds; Receptors, Antigen, T-Cell, gamma-delta; T-Lymphocyte Subsets; Zoledronic Acid

Human Vgamma2Vdelta2 T cells recognize nonpeptide antigens, such as isoprenoid pyrophosphomonoester intermediates, alkylamine compounds, and bisphosphonate drugs, as well as some tumor cells. Although attempts have been made to derive novel cancer immunotherapies based on the discovery of these unconventional antigens, effective therapies remain to be developed. Here, we synthesized a series of pyrophosphate-containing compounds and examined the chemical requirements for the recognition of pyrophosphomonoester antigens by gammadelta T cells. The structural analysis clearly demonstrated that a proximal methylene moiety plays a crucial role in the stimulatory activity of the antigens. For optimal gammadelta T cell proliferation, we find that the use of human serum albumin was preferred and that pyrophosphomonoesters were superior to nitrogen-containing bisphosphonate compounds. Using these techniques, we have successfully expanded gammadelta T cells from healthy donors as well as from cancer patients using one of the most active compounds, 2-methyl-3-butenyl-1-pyrophosphate (2M3B1PP). The resulting expanded gammadelta T cells exhibited potent, cytotoxic activity against a wide variety of tumor cell lines. Even gammadelta T cells from a patient with advanced liver carcinoma efficiently responded to 2M3B1PP and exhibited strong cytotoxic activity against tumor cells. The pretreatment of tumor cells with nonpeptide antigens was essential for efficient cytotoxicity via TCR-gammadelta. The present study suggests a novel strategy for cancer immunotherapy using synthetic small pyrophosphate-containing compounds and nitrogen-containing bisphosphonates.

Topics: Antibody Specificity; Antigens, Neoplasm; Antineoplastic Agents; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Clone Cells; Diphosphates; Diphosphonates; Flow Cytometry; Humans; Immunotherapy; Interleukin-2; Jurkat Cells; Models, Molecular; Monocytes; Neoplasms; T-Lymphocytes

Sequential liver scintiphotography with Tc-99m pyrophosphate (PYP) was used to prospectively evaluate its uptake patterns in hepatoma. The scintiphotos and time-activity curves of 40 cases were analyzed. Two distinct patterns of tumor activity were noted: gradual but complete extraction and trapping of Tc-99m PYP in hepatoma in 38% of the patients (group 1), and absence of subsequent Tc-99m PYP uptake in hepatoma after initial blood pool activity in 62% of the patients (group 2). Since extraction and trapping of Tc-99m PYP occur approximately in two fifths of the patients with hepatoma, we conclude that Tc-99m PYP liver scintigraphy is not worthwhile supplementing the conventional radionuclide studies for diagnosing hepatoma, even in the selected patients in the countries where the prevalence of hepatoma is high.

Topics: Carcinoma, Hepatocellular; Diphosphates; Humans; Liver; Liver Neoplasms; Prospective Studies; Radionuclide Imaging; Technetium; Technetium Tc 99m Pyrophosphate; Technetium Tc 99m Sulfur Colloid; Time Factors

Previously, we reported that the rate of metabolism of methyl sterol intermediates of cholesterol biosynthesis by broken-cell preparations of Morriss hepatoma 7777 is very slow, whereas the intact tumors are known to synthesize cholesterol quite efficiently. Active preparations have now been obtained by substitution of pyrophosphate for phosphate buffer. Although substitution of pyrophosphate buffer markedly enhances microsomal methyl sterol demethylation rates 3- to 4-fold in hepatoma 7777, other microsomal enzymes and electron carriers in either liver or a more slowly growing hepatoma appear to be unaffected by pyrophosphate. Several properties of the active microsomal methyl sterol demethylase have now been compared for control rat liver, host liver, tumor 7777, and tumor 5123C. Conditions necessary for the assay of initial velocities of enzymic reactions in the tumor microsomes have been established with respect to the amount of protein, time-course, concentrations of cofactors and substrate, pH, and other variables. The K'm and the responses to the variables studied above are very similar for methyl sterol demethylase of microsomes isolated from control liver, host liver, tumor 5123C, and tumor 7777. The multienzymic demethylase in the various preparations has been found to be inhibited similarly by in vitro additions of cyanide, cytochrome c, and bile salts. Thus, the enzymes of the microsomal-bound 4-methyl sterol demethylase of cholesterol biosynthesis appear to be very similar in liver and these 2 Morris hepatomas. When xenobiotic inducers of microsomal oxidases, such as phenobarbital and methylcholanthrene, are administered to normal and tumor-bearing rats, elevated rates of methyl sterol demethylation are observed with isolated liver microsomes obtained from both normal and tumor-bearing rats. Similar increases are not observed in the tumors. Furthermore, daily administration of an intestinal bile acid sequestrant elevates hepatic methyl sterol demethylase, but statistically significant changes were not observed in tumors 7777 and 5123C. Since the enzymes of methyl sterol demethylase appear to be grossly similar in liver and these hepatomas, regulation of the activity of the multienzymic system contained in the tumors may be altered. On the other hand, these agents in vivo simply may not affect liver and the hepatomas similarly, due to a lack of uptake of the foreign substances by the tumor that has been transplanted to the thighs.

Topics: Animals; Buffers; Carcinoma, Hepatocellular; Cholesterol; Cholestyramine Resin; Cyanides; Cytochrome c Group; Diphosphates; Edetic Acid; Liver; Liver Neoplasms; Male; Methylcholanthrene; Microsomes, Liver; NADH, NADPH Oxidoreductases; Neoplasms, Experimental; Phenobarbital; Phosphates; Rats; Steroid Hydroxylases; Sterols

Topics: Animals; Carcinoma, Hepatocellular; Cell Line; Cells, Cultured; Cycloheximide; Cytidine; Deamination; Deoxyadenosines; Deoxyribonucleosides; Deoxyuridine; Diphosphates; Dipyridamole; DNA, Neoplasm; Guanine Nucleotides; Hydroxyurea; Hypoxanthines; Inosine; Inosine Nucleotides; Liver Neoplasms; Neoplasms, Experimental; Phosphates; Rats; RNA, Neoplasm; Thymidine; Time Factors; Tritium

Topics: Animals; Carbon Isotopes; Carcinoma, Hepatocellular; Centrifugation, Density Gradient; Cholesterol; Citrates; Diphosphates; Electrophoresis, Polyacrylamide Gel; Half-Life; Leucine; Liver; Liver Neoplasms; Male; Methods; Microsomes, Liver; Molecular Weight; Neoplasm Proteins; Neoplasms, Experimental; Phospholipids; Polytetrafluoroethylene; Proteins; Rats; Sodium Dodecyl Sulfate; Urea

Topics: Adenosine Triphosphate; Amino Acids; Animals; Ascitic Fluid; Carbon Radioisotopes; Carcinoma, Hepatocellular; Cell Nucleolus; Centrifugation, Density Gradient; Cytoplasm; Diphosphates; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Neoplasm Proteins; Neoplasms, Experimental; Phosphates; Phosphorus Radioisotopes; Rats; RNA, Transfer; Transfer RNA Aminoacylation

Topics: Aging; Animals; Animals, Newborn; Carbon Isotopes; Carcinoma, Hepatocellular; Cytoplasm; Diphosphates; Liver; Liver Neoplasms; Male; Neoplasms, Experimental; Nucleic Acids; Orotic Acid; Pentosyltransferases; Rats; Ribose; Thymidine; Tritium; Uridine

A system for study and measurement of the attachment in vitro of exogenous polyribosomes to membranes has been presented. Its main features are use of low temperature, post-microsomal supernatant, pyrophosphate and citric acid to remove ribosomes from the surface of rough endoplasmic reticulum, and a method for quantitative separation of unattached from membrane-associated polyribosomes. The following were found. (1) Rough endoplasmic reticulum, from which ribosomes had been removed by treatment with pyrophosphate and citrate, bound over 50% of added polyribosomes, whereas the untreated (or control) rough and smooth endoplasmic reticulum and the smooth endoplasmic reticulum treated with pyrophosphate-citrate did not bind polyribosomes. (2) The polyribosome-binding capacity of rough endoplasmic reticulum stripped of its ribosomes decayed upon storage of the membranes at 0-4 degrees C. The half-life of this decay was about 6 days whereas that of the polyribosome-binding capacity of hepatoma stripped rough endoplasmic reticulum was about 1.5 days. (3) Preparations of stripped rough endoplasmic reticulum after reassociation with polyribosomes in vitro were quite similar to preparations of native rough endoplasmic reticulum as viewed with the electron microscope. Evidence is presented to support the contention that association of polyribosomes with membranes was the result of polyribosomal reattachment to the membranes rather than trapping of the polyribosomes between vesicles of the membranes.

Topics: Animals; Carcinoma, Hepatocellular; Citrates; Diphosphates; Endoplasmic Reticulum; In Vitro Techniques; Liver; Liver Neoplasms; Male; Methods; Microscopy, Electron; Microsomes, Liver; Neoplasms, Experimental; Phosphorus Isotopes; Rats; Ribosomes; Temperature

Topics: Adenine Nucleotides; Adenosine Triphosphate; Ammonium Chloride; Animals; Asparagine; Aspartic Acid; Carbon Isotopes; Carcinoma, Hepatocellular; Diphosphates; Female; Glutamine; Hydrogen-Ion Concentration; Hydroxamic Acids; Hydroxylamines; Liver Neoplasms; Magnesium; Manganese; Neoplasms, Experimental; Potassium; Rats

Topics: Animals; Bile Acids and Salts; Carcinoma, Hepatocellular; Cell Fractionation; Cell Line; Centrifugation, Density Gradient; Centrifugation, Zonal; Culture Techniques; Diphosphates; Encephalomyocarditis virus; Liver Neoplasms; Microsomes; Mitochondria; Rats; Ribonucleases; RNA Nucleotidyltransferases; RNA, Viral; Temperature; Time Factors

Topics: Carbon Isotopes; Carcinoma, Hepatocellular; Chemistry Techniques, Analytical; Choline; Cytosine Nucleotides; Deoxycytosine Nucleotides; Diphosphates; Lecithins; Lipid Metabolism; Liver; Liver Neoplasms; Neoplasms, Experimental; Phosphatidylcholines; Rats; Research; Ribonucleases