piperidines and 23-hydroxybetulinic-acid

Hepatocellular carcinoma (HCC) shows recurrence and lung metastasis even after treatment. 23-hydroxybetulinic acid (23-HBA), a major active constituent of Pulsatilla chinensis, exhibits potent antitumor activities. We herein investigate the biological effect of 23-HBA on metastasis and immunosuppression in a mouse model of HCC. Microarray-based gene expression profiling was employed to identify the target genes of 23-HBA in the treatment of HCC. The effect of 23-HBA on the progression of HCC was evaluated by in-vitro cell function measurements along with in-vivo xenograft implantation, lung metastasis and CD11b+Gr1+ staining experiments. The potential mechanism involving target signaling pathway was investigated by western blot analysis. Bioinformatics analysis revealed that matrix metalloproteinase 2 (MMP2) was a key target gene mediated by 23-HBA in HCC, whereas Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis demonstrated that MMP2 mainly affects the development and metastasis of HCC. 23-HBA significantly reduced cell malignant functions in vitro while delaying the HCC growth and metastasis in vivo. In addition, the number of myeloid-derived suppressor cells was shown to be reduced following administration of 23-HBA in mice. Mechanistic analysis indicated that these effects of 23-HBA during HCC were involved with the mitogen-activated protein kinase (MAPK) signaling pathway inactivation and resulted in decreased phosphorylation of both mitogen-activated protein kinases 1/2 and extracellular signal-regulated kinase 1/2. Our study reveals that 23-HBA acts as a tumor suppressor agent and suppresses HCC tumorigenesis, metastasis and immunosuppression via blockade of the MAPK signaling pathway, suggesting that 23-HBA may serve as a promising drug target to treat HCC.

Topics: Animals; Carcinogenesis; Carcinoma, Hepatocellular; Cell Line, Tumor; Cell Proliferation; Disease Models, Animal; Gene Expression Regulation, Neoplastic; Humans; Immunosuppression Therapy; Liver Neoplasms; Lung Neoplasms; Matrix Metalloproteinase 2; Mice; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinases; Piperidines; Signal Transduction; Triterpenes

Multidrug resistance (MDR) is a major obstacle to successful chemotherapy for cancer; thus, novel MDR reversers are urgently needed. In the present study, we assessed whether two synthetic derivatives of 23-hydroxybetulinic acid, 3,23-O-diacetyl-17-1,4'-bipiperidinyl betulinic amide (DABB) and 3,23-O-dihydroxy-17-1,4'-bipiperidinyl betulinic amide (DHBB), could reverse MDR induced by ATP-binding cassette (ABC) transporters. Using the MTT assay, we found that DABB and DHBB could enhance the cytotoxicities of ABCB1 substrates doxorubicin, vincristine, and paclitaxel in ABCB1-overexpressing HepG2/ADM and MCF-7/ADR cells, whereas that of a non-ABCB1 substrate cisplatin was unaffected. The ABCB1 substrate accumulation and efflux assay showed that DABB and DHBB not only enhanced the retention of doxorubicin and rhodamine123 but also inhibited the efflux of rhodamine123. Further mechanistic studies by reverse transcription PCR, western blot, and ABCB1 ATPase activity assay indicated that DABB and DHBB suppressed ABCB1 ATPase activity, but did not alter mRNA or protein expression of ABCB1. ABCB1 siRNA pretreatment attenuated the reversal effect of DABB and DHBB, indicating that their reversal effects were partially dependent on ABCB1. Docking analysis also implied that DABB and DHBB bind directly to ABCB1 at a site partly overlapped with that of verapamil. Taken together, our findings suggest that two bipiperidinyl derivatives of 23-hydroxybetulinic acid reverse ABCB1-mediated MDR through modulation of ABCB1 ATPase activity, thereby inhibiting its efflux function in both HepG2/ADM and MCF-7/ADR cells. These findings may contribute toward the development of novel MDR reversers using DABB and DHBB as adjuvant anticancer chemotherapy.

Topics: Antineoplastic Agents; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Cell Line, Tumor; Cytochrome P-450 CYP3A; Cytochrome P-450 CYP3A Inhibitors; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Drug Screening Assays, Antitumor; Hep G2 Cells; Humans; MCF-7 Cells; Molecular Docking Simulation; Paclitaxel; Piperidines; Rhodamine 123; Triterpenes; Verapamil; Vincristine

23-O-(1,4'-Bipiperidine-1-carbonyl)betulinic acid (BBA), a synthetic derivative of 23-hydroxybetulinic acid (23-HBA), shows a reversal effect on multidrug resistance (MDR) in our preliminary screening. Overexpression of ATP-binding cassette (ABC) transporters such as ABCB1, ABCG2, and ABCC1 has been reported in recent studies to be a major factor contributing to MDR. Our study results showed that BBA enhanced the cytotoxicity of ABCB1 substrates and increased the accumulation of doxorubicin or rhodamine123 in ABCB1 overexpressing cells, but had no effect on non ABCB1 substrate, such as cisplatin; what's more, BBA slightly reversed ABCG2-mediated resistance to SN-38, but did not affect the ABCC1-mediated MDR. Further studies on the mechanism indicated that BBA did not alter the expression of ABCB1 at mRNA or protein levels, but affected the ABCB1 ATPase activity by stimulating the basal activity at lower concentrations and inhibiting the activity at higher concentrations. In addition, BBA inhibited the verapamil-stimulated ABCB1 ATPase activity and the photolabeling of ABCB1 with [(125)I] iodoarylazidoprazosin in a concentration-dependent manner, indicating that BBA directly interacts with ABCB1. The docking study confirmed this notion that BBA could bind to the drug binding site(s) on ABCB1, but its binding position was only partially overlapping with that of verapamil or iodoarylazidoprazosin. Importantly, BBA increased the inhibitory effect of paclitaxel in ABCB1 overexpressing KB-C2 cell xenografts in nude mice. Taken together, our findings suggest that BBA can reverse ABCB1-mediated MDR by inhibiting its efflux function of ABCB1, which supports the development of BBA as a novel potential MDR reversal agent used in the clinic.

Topics: Animals; Antibiotics, Antineoplastic; Antineoplastic Agents; Antineoplastic Agents, Phytogenic; Apoptosis; ATP Binding Cassette Transporter, Subfamily B; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Western; Breast Neoplasms; Calcium Channel Blockers; Camptothecin; Carcinoma, Hepatocellular; Cell Proliferation; Cells, Cultured; Cisplatin; Doxorubicin; Drug Resistance, Multiple; Drug Resistance, Neoplasm; Female; Fluorescent Antibody Technique; Humans; In Vitro Techniques; Irinotecan; KB Cells; Liver Neoplasms; Male; Mice; Mice, Nude; Models, Molecular; Molecular Docking Simulation; Paclitaxel; Piperidines; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Triterpenes; Verapamil

The mitochondrion plays a crucial role in the process of apoptosis and has thus become one of the targets for the search for potential chemotherapeutic agents. Betulinic acid [3beta-hydroxy-lup-20(19)lupaen-28-carbonic acid], a lupane-type triterpene which is abundant in many plant species, has been shown to exert a direct effect on the mitochondria and subsequent apoptosis in melanoma cells. Chemical synthesis and modification of betulinic acid are being explored to develop more potent derivatives. We present here the apoptotic activity of several natural derivatives of betulinic acid which were isolated from the roots of a Chinese medicinal herb, Pulsatilla chinensis (Bge) Regel [Ye, W., Ji, N.N., Zhao, S.X., Liu, J.H., Ye, T., McKervey, M.A., Stevenson, P., 1996. Triterpenoids from Pulsatilla chinensis. Phytochemistry 42, 799-802]. Of the five compounds tested, 3-oxo-23-hydroxybetulinic acid was the most cytotoxic on murine melanoma B16 cells (IC50=22.5 microg/ml), followed by 23-hydroxybetulinic acid and betulinic acid (IC50=32 and 76 microg/ml, respectively), with lupeol and betulin exhibiting the weakest cytotoxicity (IC50> or =100 microg/ml). Exposure of B16 cells to betulinic acid, 23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid caused a rapid increase in reactive oxidative species production and a concomitant dissipation of mitochondrial membrane potential in a dose- and time-dependent manner, which resulted in cell apoptosis, as demonstrated by fluorescence microscopy, gel electrophoresis and flow-cytometric analysis. Cell cycle analysis further demonstrated that both 3-oxo-23-hydroxybetulinic acid and 23-hydroxybetulinic acid dramatically increased DNA fragmentation at the expense of G1 cells at doses as low as 12.5 and 25 microg/ml, respectively, thereby showing their potent apoptotic properties. Our results showed that hydroxylation at the C3 position of betulinic acid is likely to enhance the apoptotic activity of betulinic acid derivatives (23-hydroxybetulinic acid and 3-oxo-23-hydroxybetulinic acid) on murine melanoma B16 cells.

Topics: Animals; Apoptosis; Betulinic Acid; Cell Cycle; Cell Line, Tumor; Cell Survival; Dose-Response Relationship, Drug; Inhibitory Concentration 50; Intracellular Membranes; Melanoma, Experimental; Membrane Potentials; Mice; Mitochondria; NADPH Oxidases; Pentacyclic Triterpenes; Piperidines; Propionates; Reactive Oxygen Species; Structure-Activity Relationship; Triterpenes