The dichloromethane fraction from Calotropis gigantea (L.) dryand. Stem bark extract prevents liver cancer in SDT rats with insulin-independent diabetes mellitus.

ETHNOPHARMACOLOGICAL RELEVANCE: Calotropis gigantea (L.) Dryand. (C. gigantea) is a traditional medicinal plant, recognized for its effectiveness in managing diabetes, along with its notable antioxidant, anti-inflammatory, and anticancer properties. Type II diabetes mellitus (T2DM) is characterized by chronic metabolic disorders associated with an elevated risk of hepatocellular carcinoma (HCC) due to hyperglycemia and impaired insulin response. The scientific validation of C. gigantea's ethnopharmacological efficacy offers advantages in alleviating cancer progression in T2DM complications, enriching existing knowledge and potentially aiding future clinical cancer treatments. AIM: This study aimed to investigate the preventive potential of the dichloromethane fraction of C. gigantea stem bark extract (CGDCM) against diethylnitrosamine (DEN)-induced HCC in T2DM rats, aiming to reduce cancer incidence associated with diabetes while validating C. gigantea's ethnopharmacological efficacy. MATERIALS AND METHODS: Spontaneously Diabetic Torii (SDT) rats were administered DEN to induce HCC (SDT-DEN-VEH), followed by treatment with CGDCM. Metformin was used as a positive control (SDT-DEN-MET). All the treatments were administered for 10 weeks after the initial DEN injection. Diabetes-related parameters, including serum levels of glucose, insulin, and glycosylated hemoglobin (HbA1c), as well as liver function enzymes (aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and gamma-glutamyl transferase), were quantified. Serum inflammation biomarkers interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were evaluated. Liver tissue samples were analyzed for inflammation protein expression (IL-6, TNF-α, transforming growth factor-ß1 (TGF-ß1), and α-smooth muscle actin (α-SMA)). Histopathological evaluation was performed to assess hepatic necrosis, inflammation, and fibrosis. Liver cell proliferation was determined using immunohistochemistry for Ki-67 expression. RESULTS: Rats with SDT-DEN-induced HCC treated with CGDCM exhibited reduced serum glucose levels, elevated insulin levels, and decreased HbA1c levels. CGDCM treatment also reduced elevated hepatic IL-6, TNF-α, TGF-ß1, and α-SMA levels in SDT-DEN-VEH rats. Additionally, CGDCM treatment prevented hepatocyte damage, fibrosis, and cell proliferation. No adverse effects on normal organs were observed with CGDCM treatment, suggesting its safety for the treatment of HCC complications associated with diabetes. Additionally, the absence of adverse effects in SD rats treated with CGDCM at 2.5 mg/kg further supports the notion of its safe usage. CONCLUSIONS: These findings suggest that C. gigantea stem bark extract exerts preventive effects against the development of HCC complications in patients with T2DM, expanding the potential benefits of its ethnopharmacological advantages.

Combination of ethyl acetate fraction from Calotropis gigantea stem bark and sorafenib induces apoptosis in HepG2 cells.

The cytotoxicity of the ethyl acetate fraction of the Calotropis gigantea (L.) Dryand. (C. gigantea) stem bark extract (CGEtOAc) has been demonstrated in many types of cancers. This study examined the improved cancer therapeutic activity of sorafenib when combined with CGEtOAc in HepG2 cells. The cell viability and cell migration assays were applied in HepG2 cells treated with varying concentrations of CGEtOAc, sorafenib, and their combination. Flow cytometry was used to determine apoptosis, which corresponded with a decline in mitochondrial membrane potential and activation of DNA fragmentation. Reactive oxygen species (ROS) levels were assessed in combination with the expression of the phosphatidylinositol-3-kinase (PI3K)/ protein kinase B (Akt)/ mammalian target of rapamycin (mTOR) pathway, which was suggested for association with ROS-induced apoptosis. Combining CGEtOAc at 400 µg/mL with sorafenib at 4 µM, which were their respective half-IC50 concentrations, significantly inhibited HepG2 viability upon 24 h of exposure in comparison with the vehicle and each single treatment. Consequently, CGEtOAc when combined with sorafenib significantly diminished HepG2 migration and induced apoptosis through a mitochondrial-correlation mechanism. ROS production was speculated to be the primary mechanism of stimulating apoptosis in HepG2 cells after exposure to a combination of CGEtOAc and sorafenib, in association with PI3K/Akt/mTOR pathway suppression. Our results present valuable knowledge to support the development of anticancer regimens derived from the CGEtOAc with the chemotherapeutic agent sorafenib, both of which were administered at half-IC50, which may minimize the toxic implications of cancer treatments while improving the therapeutic effectiveness toward future medical applications.

Enhanced apoptosis of HCT116 colon cancer cells treated with extracts from Calotropis gigantea stem bark by starvation.

Calotropis gigantea stem bark extract, particularly the dichloromethane fraction (CGDCM), demonstrated the most potent antiproliferative effects on hepatocellular carcinoma HepG2 and colorectal HCT116 cells. The current study focused on enhancing the effectiveness of cancer treatment with CGDCM at concentrations close to the IC50 in HCT116 cells by reducing their nutrient supply. CGDCM (2, 4, and 8 µg/mL) treatment for 24 h under glucose conditions of 4.5 g/L without fetal bovine serum (FBS) supplementation or serum starvation (G+/F-), glucose 0 g/L with 10% FBS or glucose starvation (G-/F+), and glucose 0 g/L with 0% FBS or complete starvation (G-/F-) induced a greater antiproliferative effect in HCT116 cells than therapy in complete medium with glucose 4.5 g/L and 10% FBS (G+/F+). Nonetheless, the anticancer effect of CGDCM at 4 µg/mL under (G-/F-) showed the highest activity compared to other starvation conditions. The three starvation conditions showed a significant reduction in cell viability compared to the control (G+/F+) medium group, while the inhibitory effect on cell viability did not differ significantly among the three starvation conditions. CGDCM at 4 µg/mL in (G-/F-) medium triggered apoptosis by dissipating the mitochondrial membrane potential and arresting cells in the G2/M phase. This investigation demonstrated that a decrease in intracellular ATP and fatty acid levels was associated with enhanced apoptosis by treatment with CGDCM at 4 µg/mL under (G-/F-) conditions. In addition, under (G-/F-), CGDCM at 4 µg/mL increased levels of reactive oxygen species (ROS) and was suggested to primarily trigger apoptosis in HCT116 cells. Thus, C. gigantea extracts may be useful for the future development of alternative, effective cancer treatment regimens.

Calotropis gigantea stem bark extract activates HepG2 cell apoptosis through ROS and its effect on cytochrome P450.

The 95% ethanolic extract of the dry powder of Calotropis gigantea (C. gigantea) stem bark was separated by fractionation with different solutions to yield 4 fractions: dichloromethane (CGDCM), ethyl acetate (CGEtOAc), and water (CGW). This research focused on CGDCM-induced apoptosis in HepG2 cells with IC50 and above-IC50 values, which provide useful information for future anticancer applications. CGDCM had lower cytotoxicity on normal lung fibroblast IMR-90 cells than on HepG2 cells. Apoptotic induction of CGDCM was mediated by decreased fatty acid and ATP synthesis while increasing reactive oxygen species production. The effects of the four extracts on the activity of the four major CYP450 isoforms (CYP1A2, CYP2C9, CYP2E1 and CYP3A4) were determined using the CYP-specific model activity of each isoform. All four fractions of the extract were shown to be poor inhibitors of CYP1A2 and CYP2E1 (IC50 > 1000 µg/mL) and moderate inhibitors of CYP3A4 (IC50 = 56.54-296.9 µg/mL). CGDCM and CGW exerted moderate inhibition activities on CYP2C9 (IC50 = 59.56 and 46.38 µg/mL, respectively), but CGEtOH and CGEtOAc exhibited strong inhibition activities (IC50 = 12.11 and 20.43 µg/mL, respectively). It is proposed that C. gigantea extracts at high doses have potential for further studies to develop alternative anticancer applications. Inhibiting CYP2C9 activity may also lead to drug-herb interactions.

Calotropis gigantea stem bark extracts inhibit liver cancer induced by diethylnitrosamine.

Several fractions of Calotropis gigantea extracts have been proposed to have potential anticancer activity in many cancer models. The present study evaluated the anticancer activity of C. gigantea stem bark extracts in liver cancer HepG2 cells and diethylnitrosamine (DEN)-induced primary liver cancer in rats. The carcinogenesis model induced by DEN administration has been widely used to study pathophysiological features and responses in rats that are comparable to those seen in cancer patients. The dichloromethane (CGDCM), ethyl acetate, and water fractions obtained from partitioning crude ethanolic extract were quantitatively analyzed for several groups of secondary metabolites and calactin contents. A combination of C. gigantea stem bark extracts with doxorubicin (DOX) was assessed in this study to demonstrate the enhanced cytotoxic effect to cancer compared to the single administration. The combination of DOX and CGDCM, which had the most potential cytotoxic effect in HepG2 cells when compared to the other three fractions, significantly increased cytotoxicity through the apoptotic effect with increased caspase-3 expression. This combination treatment also reduced ATP levels, implying a correlation between ATP and apoptosis induction. In a rat model of DEN-induced liver cancer, treatment with DOX, C. gigantea at low (CGDCM-L) and high (CGDCM-H) doses, and DOX + CGDCM-H for 4 weeks decreased the progression of liver cancer by lowering the liver weight/body weight ratio and the occurrence of liver hyperplastic nodules, fibrosis, and proliferative cells. The therapeutic applications lowered TNF-α, IL-6, TGF-ß, and α-SMA inflammatory cytokines in a similar way, implying that CGDCM had a curative effect against the inflammation-induced liver carcinogenesis produced by DEN exposure. Furthermore, CGDCM and DOX therapy decreased ATP and fatty acid synthesis in rat liver cancer, which was correlated with apoptosis inhibition. CGDCM reduced cleaved caspase-3 expression in liver cancer rats when used alone or in combination with DOX, implying that apoptosis-inducing hepatic carcinogenesis was suppressed. Our results also verified the low toxicity of CGDCM injection on the internal organs of rats. Thus, this research clearly demonstrated a promising, novel anticancer approach that could be applied in future clinical studies of CGDCM and combination therapy.

Epigallocatechin gallate triggers apoptosis by suppressing de novo lipogenesis in colorectal carcinoma cells.

The de novo lipogenesis (DNL) pathway has been identified as a regulator of cancer progression and aggressiveness. Downregulation of key lipogenesis enzymes has been shown to activate apoptosis in cancerous cells. Epigallocatechin gallate (EGCG) inhibits cancer cell proliferation without causing cytotoxicity in healthy cells. The present study aimed to investigate the effects of EGCG on the promotion of apoptosis associated with the DNL pathway inhibition in cancer cells, both in vitro and in vivo. We observed that two colorectal cancer cell lines (HCT116 and HT-29) had a higher cytotoxic response to EGCG treatment than hepatocellular carcinoma cells, including HepG2 and HuH-7. EGCG treatment decreased cell viability and increased mitochondrial damage-triggered apoptosis in both HCT116 and HT-29 cancer cells. Additionally, we treated mice transplanted with HCT116 cells with 30 or 50 mg·kg-1 EGCG for 7 days to evaluate the apoptotic effects of EGCG treatment in a xenograft mouse model of cancer. We observed a decrease in intracellular fatty acid levels, which suggested that EGCG-induced apoptosis was associated with a decrease in fatty acid levels in cancer. Suppression of ATP synthesis by EGCG indicated that cell death induction in cancer cells could be mediated by shared components of the DNL and energy metabolism pathways. In addition, EGCG-induced apoptosis suppressed the expression of the phosphorylation protein kinase B and extracellular signal-regulated kinase 1/2 signaling proteins in tumors from xenografted mice. Cytotoxic effects in unaffected organs and tissues of the mouse xenograft model were absent upon EGCG treatment.

Calotropis gigantea stem bark extract induced apoptosis related to ROS and ATP production in colon cancer cells.

Conventional chemotherapeutic agents for colorectal cancer (CRC) cause systemic side effects and eventually become less efficacious owing to the development of drug resistance in cancer cells. Therefore, new therapeutic regimens have focused on the use of natural products. The anticancer activity of several parts of Calotropis gigantea has been reported; however, the effects of its stem bark extract on inhibition of cancer cell proliferation have not yet been examined. In this study, the anticancer activity of C. gigantea stem bark extract, both alone and in combination with 5-fluorouracil (5-FU), was evaluated. A crude ethanolic extract was prepared from dry, powdered C. gigantea barks using 95% ethanol. This was then partitioned to obtain dichloromethane (CGDCM), ethyl acetate, and water fractions. Quantitative analysis of the constituent secondary metabolites and calotropin was performed. These fractions exhibited cytotoxicity in HCT116 and HT-29 cells, with CGDCM showing the highest potency in both the cell lines. A combination of CGDCM and 5-FU significantly enhanced the cytotoxic effect. Moreover, the resistance of normal fibroblast, HFF-1, cells to this combination demonstrated its safety in normal cells. The combination significantly enhanced apoptosis through the mitochondria-dependent pathway. Additionally, the combination reduced adenosine triphosphate production and increased the production of reactive oxygen species, demonstrating the mechanisms involved in the induction of apoptosis. Our results suggest that CGDCM is a promising anti-cancer agent and may enhance apoptosis induction by 5-FU in the treatment of CRC, while minimizing toxicity toward healthy cells.

Aphrodisiac Activity of Eulophia macrobulbon Extract on Erectile Dysfunction in Male Aged Rats.

This study investigated the effect of Eulophia macrobulbon (EM) extract on sexual performance in aged-related erectile dysfunction (ED) rats. The ethanol EM extract at the doses of 15, 150, and 450 and sildenafil citrate at the dose of 5 mg/kg body weight (BW) were administered orally to the aged male rats once daily for 21 days. Mating parameters and intracavernosal pressure (ICP) were measured to evaluate their sexual and erection functions. Numbers of sperm and sperm motility as well as the diameter of seminiferous tubules were observed. The serum testosterone and 3',5'-cyclic guanosine monophosphate (cGMP) concentration in the rat penile tissue were analyzed. The results showed the significant increased sexual motivation, copulatory performance, and ICP of aged rats treated with sildenafil citrate and all doses of EM extract as compared to control aged rats. Moreover, their serum testosterone levels were slightly increased and significant increase in penile cGMP concentration was observed in these aged rats treated with sildenafil citrate and EM extract. The results suggest that treatment with EM could inhibit activity of PDE5 in penile tissue resulting in the increased cGMP level and bring to the improvement of erectile function and sexual performance.

Suppressed de novo lipogenesis by plasma membrane citrate transporter inhibitor promotes apoptosis in HepG2 cells.

Suppression of the expression or activities of enzymes that are involved in the synthesis of de novo lipogenesis (DNL) in cancer cells triggers cell death via apoptosis. The plasma membrane citrate transporter (PMCT) is the initial step that translocates citrate from blood circulation into the cytoplasm for de novo long-chain fatty acids synthesis. This study investigated the antitumor effect of the PMCT inhibitor (PMCTi) in inducing apoptosis by inhibiting the DNL pathway in HepG2 cells. The present findings showed that PMCTi reduced cell viability and enhanced apoptosis through decreased intracellular citrate levels, which consequently caused inhibition of fatty acid and triacylglycerol productions. Thus, as a result of the reduction in fatty acid synthesis, the activity of carnitine palmitoyl transferase-1 (CPT-1) was suppressed. Decreased CPT-1 activity also facilitated the disruption of mitochondrial membrane potential (ΔΨm) leading to stimulation of apoptosis. Surprisingly, primary human hepatocytes were not affected by PMCTi. Increased caspase-8 activity as a consequence of reduction in fatty acid synthesis was also found to cause disruption of ΔΨm. In addition, apoptosis induction by PMCTi was associated with an enhanced reactive oxygen species generation. Taken together, we suggest that inhibition of the DNL pathway following reduction in citrate levels is an important regulator of apoptosis in HepG2 cells via suppression of CPT-1 activity. Thus, targeting the DNL pathway mediating CPT-1 activity by PMCTi may be a selective potential anticancer therapy.

Combination of Mitochondrial and Plasma Membrane Citrate Transporter Inhibitors Inhibits De Novo Lipogenesis Pathway and Triggers Apoptosis in Hepatocellular Carcinoma Cells.

Increased expression levels of both mitochondrial citrate transporter (CTP) and plasma membrane citrate transporter (PMCT) proteins have been found in various cancers. The transported citrates by these two transporter proteins provide acetyl-CoA precursors for the de novo lipogenesis (DNL) pathway to support a high rate of cancer cell viability and development. Inhibition of the DNL pathway promotes cancer cell apoptosis without apparent cytotoxic to normal cells, leading to the representation of selective and powerful targets for cancer therapy. The present study demonstrates that treatments with CTP inhibitor (CTPi), PMCT inhibitor (PMCTi), and the combination of CTPi and PMCTi resulted in decreased cell viability in two hepatocellular carcinoma cell lines (HepG2 and HuH-7). Treatment with citrate transporter inhibitors caused a greater cytotoxic effect in HepG2 cells than in HuH-7 cells. A lower concentration of combined CTPi and PMCTi promotes cytotoxic effect compared with either of a single compound. An increased cell apoptosis and an induced cell cycle arrest in both cell lines were reported after administration of the combined inhibitors. A combination treatment exhibits an enhanced apoptosis through decreased intracellular citrate levels, which consequently cause inhibition of fatty acid production in HepG2 cells. Apoptosis induction through the mitochondrial-dependent pathway was found as a consequence of suppressed carnitine palmitoyl transferase-1 (CPT-1) activity and enhanced ROS generation by combined CTPi and PMCTi treatment. We showed that accumulation of malonyl-CoA did not correlate with decreasing CPT-1 activity. The present study showed that elevated ROS levels served as an inhibition on Bcl-2 activity that is at least in part responsible for apoptosis. Moreover, inhibition of the citrate transporter is selectively cytotoxic to HepG2 cells but not in primary human hepatocytes, supporting citrate-mediating fatty acid synthesis as a promising cancer therapy.

Epistructured catechins, EGCG and EC facilitate apoptosis induction through targeting de novo lipogenesis pathway in HepG2 cells.

BACKGROUND: Abnormally high expression of the mammalian de novo lipogenesis (DNL) pathway in various cancer cells promotes cell over-proliferation and resistance to apoptosis. Inhibition of key enzymes in the DNL pathway, namely, ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase (FASN) can increase apoptosis without cytotoxicity to non-cancerous cells, leading to the search for and presentation of novel selective and powerful targets for cancer therapy. Previous studies reported that epistructured catechins, epigallocatechin gallate (EGCG) and epicatechin (EC) exhibit different mechanisms regarding a strong inducer of apoptosis in various cancer cell lines. Thus, the current study investigated the growth inhibitory effect of EGCG and EC, on the enzyme expression and activity of the DNL pathway, which leads to the prominent activity of carnitine palmitoyl transferase-1 (CPT-1) mediating apoptosis in HepG2 cells. METHODS: The cytotoxicity on HepG2 cells of EGCG and EC was determined by MTT assay. Cell death caused by apoptosis, the dissipation of mitochondrial membrane potential (MMP), and cell cycle arrest were then detected by flow cytometry. We further investigated the decrease of fatty acid levels associated with DNL retardation, followed by evaluation of DNL protein expression. Then, the negative inhibitory effect of depleted fatty acid synthesis on malonyl-CoA synthesis followed by regulating of CPT-1 activity was investigated. Thereafter, we inspected the enhanced reactive oxygen species (ROS) generation, which is recognized as one of the causes of apoptosis in HepG2 cells. RESULTS: We found that EGCG and EC decreased cancer cell viability by increasing apoptosis as well as causing cell cycle arrest in HepG2 cells. Apoptosis was associated with MMP dissipation. Herein, EGCG and EC inhibited the expression of FASN enzymes contributing to decreasing fatty acid levels. Notably, this decrease consequently showed a suppressing effect on the CPT-1 activity. We suggest that epistructured catechin-induced apoptosis targets CPT-1 activity suppression mediated through diminishing the DNL pathway in HepG2 cells. In addition, increased ROS production was found after treatment with EGCG and EC, indicating oxidative stress mechanism-induced apoptosis. The strong apoptotic effect of EGCG and EC was specifically absent in primary human hepatocytes. CONCLUSION: Our supportive evidence confirms potential alternative cancer treatments by EGCG and EC that selectively target the DNL pathway.

[6]-Gingerol inhibits de novo fatty acid synthesis and carnitine palmitoyltransferase-1 activity which triggers apoptosis in HepG2.

The de novo fatty acid synthesis catalyzed by key lipogenic enzymes, including fatty acid synthase (FASN) has emerged as one of the novel targets of anti-cancer approaches. The present study explored the possible inhibitory efficacy of [6]-gingerol on de novo fatty acid synthesis associated with mitochondrial-dependent apoptotic induction in HepG2 cells. We observed a dissipation of mitochondrial membrane potential accompanied by a reduction of fatty acid levels. [6]-gingerol administration manifested inhibition of FASN expression, indicating FASN is a major target of [6]-gingerol inducing apoptosis in HepG2 cells. Indeed, we found that increased ROS generation could likely be a mediator of the anti-cancer effect of [6]-gingerol. A reduction of fatty acid levels and induction of apoptosis were restored by inhibition of acetyl-CoA carboxylase (ACC) activity, suggesting an accumulation of malonyl-CoA level could be the major cause of apoptotic induction of [6]-gingerol in HepG2 cells. The present study also showed that depletion of fatty acid following [6]-gingerol treatment caused an inhibitory effect on carnitine palmitoyltransferase-1 activity (CPT-1), whereas C75 augmented CPT-1 activity, indicating that [6]-gingerol exhibits the therapeutic benefit on suppression of fatty acid ß-oxidation.

The selective target of capsaicin on FASN expression and de novo fatty acid synthesis mediated through ROS generation triggers apoptosis in HepG2 cells.

The inhibition of the mammalian de novo synthesis of long-chain saturated fatty acids (LCFAs) by blocking the fatty acid synthase (FASN) enzyme activity in tumor cells that overexpress FASN can promote apoptosis, without apparent cytotoxic to non-tumor cells. The present study aimed to focus on the potent inhibitory effect of capsaicin on the fatty acid synthesis pathway inducing apoptosis of capsaicin in HepG2 cells. The use of capsaicin as a source for a new FASN inhibitor will provide new insight into its possible application as a selective anti-cancer therapy. The present findings showed that capsaicin promoted apoptosis as well as cell cycle arrest in the G0/G1 phase. The onset of apoptosis was correlated with a dissipation of mitochondrial membrane potential (ΔΨm). Apoptotic induction by capsaicin was mediated by inhibition of FASN protein expression which was accompanied by decreasing its activity on the de novo fatty acid synthesis. The expression of FASN was higher in HepG2 cells than in normal hepatocytes that were resistant to undergoing apoptosis following capsaicin administration. Moreover, the inhibitory effect of capsaicin on FASN expression and activity was found to be mediated by an increase of intracellular reactive oxygen species (ROS) generation. Treatment of HepG2 cells with capsaicin failed to alter ACC and ACLY protein expression, suggesting ACC and ACLY might not be the specific targets of capsaicin to induce apoptosis. An accumulation of malonyl-CoA level following FASN inhibition represented a major cause of mitochondrial-dependent apoptotic induction instead of deprivation of fatty acid per se. Here, we also obtained similar results with C75 that exhibited apoptosis induction by reducing the levels of fatty acid without any change in the abundance of FASN expression along with increasing ROS production. Collectively, our results provide novel evidence that capsaicin exhibits a potent anti-cancer property by targeting FASN protein in HepG2 cells.

Modulation of succinate transport in Hep G2 cell line by PKC.

The cellular uptake of the tricarboxylic acid cycle (TCA) intermediates is very important for cellular metabolism. However, the transport pathways for these intermediates in liver cells are not well characterized. We have examined the transport of succinate and citrate in the human hepatoma cell line Hep G2 and found that it exhibited a higher rate of succinate compared to citrate transport, which was sodium dependent. Comparison of the transport properties of Hep G2 to that of human retinal pigment epithelial (HRPE) cells transfected with human sodium dicarboxylate transporters, hNaDC-1, hNaDC-3, and hNaCT indicated that Hep G2 cells express a combination of hNaDC-3 and hNaCT. Short period activation of protein kinase C (PKC) by phorbol 12-myristate, 13-acetate (PMA) and alpha-adrenergic receptor agonist, phenylephrine (PE), downregulated sodium-dependent succinate transport presumably via hNaDC-3. The inhibition by PMA was partially prevented by cytochalasin D, suggesting that PKC reduces the hNaDC-3 activity, at least in part, by increased endocytosis. In contrast, activation of PKA by both forskolin and epidermal growth factor (EGF) had no effect on succinate transport. Our results suggest that Hep G2 cells provide a useful model for studies of di- and tricarboxylate regulation of human liver.