The mechanism of the cytotoxic effect of Panax notoginseng extracts on prostate cancer cells.

INTRODUCTION: Panax notoginseng (Burkill) F.H. commonly referred to as Sanqi, is a Chinese herb that has long been used to treat various conditions including blood disorders and cardiovascular diseases. While Panax notoginseng has been used as an anti-cancer medicinal herb in recent years, how it achieves this therapeutic effect has not been thoroughly elucidated. The purpose of this study was to reveal more about the mechanism of the cytotoxic effect of Panax notoginseng on prostate cancer (PCa) cells. METHODS: Ethanol extract of Panax notoginseng root was authenticated using high-performance liquid chromatography (HPLC). The cytotoxic activity of this herb against PCa cells was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, flow cytometry, and enzyme-linked immunosorbent assay (ELISA). RESULTS: The assessment of cellular metabolic activity demonstrated that Panax notoginseng reduces the viability of LNCaP and 22Rv1 cells in a dose-dependent manner. Annexin-V binding flow cytometry assay showed that Panax notoginseng induces apoptosis in PCa cells. Cell cycle analysis by quantification of DNA content using flow cytometry showed that Panax notoginseng arrests the cell cycle at the G2/M phase in both LNCaP and 22Rv1 cells. Moreover, ELISA demonstrated that Panax notoginseng-treated PCa cells secrete significantly less tumor-promoting cytokine interleukin-4 (IL-4) to the supernatant compared with controls. CONCLUSIONS: These results provide evidence for the cytotoxic effects of Panax notoginseng on PCa cell lines. This botanical is a promising candidate for the complementary and integrative medicine treatment of PCa and further studies are indicated to determine the anti-cancer mechanism of Panax notoginseng.

Adenosine 3',5'-cyclic monophosphate (cAMP)-dependent phosphoregulation of mitochondrial complex I is inhibited by nucleoside reverse transcriptase inhibitors.

Nucleoside analog reverse transcriptase inhibitors (NRTIs) are known to directly inhibit mitochondrial complex I activity as well as various mitochondrial kinases. Recent observations that complex I activity and superoxide production are modulated through cAMP-dependent phosphorylation suggests a mechanism through which NRTIs may affect mitochondrial respiration via kinase-dependent protein phosphorylation. In the current study, we examine the potential for NRTIs to inhibit the cAMP-dependent phosphorylation of complex I and the associated NADH:CoQ oxidoreductase activities and rates of superoxide production using HepG2 cells. Phosphoprotein staining of immunocaptured complex I revealed that 3'-azido-3'-deoxythymidine (AZT; 10 and 50 microM), AZT monophosphate (150 microM), and 2',3'-dideoxycytidine (ddC; 1 microM) prevented the phosphorylation of the NDUFB11 subunit of complex I. This was associated with a decrease in complex I activity with AZT and AZT monophosphate only. In the presence of succinate, superoxide production was increased with 2',3'-dideoxyinosine (ddI; 10 microM) and ddC (1 microM). In the presence of succinate+cAMP, AZT showed an inverse dose-dependent effect on superoxide production. None of the NRTIs examined inhibit PKA activity suggesting that the observed effects are due to a direct interaction with complex I. These data demonstrate a direct effect of NRTIs on cAMP-dependent regulation of mitochondrial bioenergetics independent of DNA polymerase-gamma activity; in the case of AZT, these observations may provide a mechanism for the observed long-term toxicity with this drug.

Absence of a universal mechanism of mitochondrial toxicity by nucleoside analogs.

Nucleoside analogs are associated with various mitochondrial toxicities, and it is becoming increasingly difficult to accommodate these differences solely in the context of DNA polymerase gamma inhibition. Therefore, we examined the toxicities of zidovudine (AZT) (10 and 50 microM; 2.7 and 13.4 microg/ml), didanosine (ddI) (10 and 50 microM; 2.4 and 11.8 microg/ml), and zalcitabine (ddC) (1 and 5 microM; 0.21 and 1.1 microg/ml) in HepG2 and H9c2 cells without the presumption of mitochondrial DNA (mtDNA) depletion. Ethidium bromide (EtBr) (0.5 microg/ml; 1.3 microM) was used as a positive control. AZT treatment resulted in metabolic disruption (increased lactate and superoxide) and increased cell mortality with decreased proliferation, while mtDNA remained unchanged or increased (HepG2 cells; 50 microM AZT). ddC caused pronounced mtDNA depletion in HepG2 cells but not in H9c2 cells and increased mortality in HepG2 cells, but no significant metabolic disruption in either cell type. ddI caused a moderate depletion of mtDNA in both cell types but showed no other effects. EtBr exposure resulted in metabolic disruption, increased cell mortality with decreased cell proliferation, and mtDNA depletion in both cell types. We conclude that nucleoside analogs display unique toxicities within and between culture models, and therefore, care should be taken when generalizing about the mechanisms of nucleoside reverse transcriptase inhibitor toxicity. Additionally, mtDNA abundance does not necessarily correlate with metabolic disruption, especially in cell culture; careful discernment is recommended in this regard.

Direct, DNA pol-gamma-independent effects of nucleoside reverse transcriptase inhibitors on mitochondrial bioenergetics.

Nucleoside reverse transcriptase inhibitor (NRTI)-induced cardiomyopathy has been suggested to reflect mitochondrial targets of drug toxicity. The prevailing hypothesis is that, through structural mimicry, the NRTIs are mistaken as substrates for DNA polymerase and incorporated into replicating DNA, where they cause truncation of the elongating strand. Although there exist five forms of nuclear DNA polymerase, mitochondria possess solely DNA polymerase-gamma (pol-gamma), which is a preferred target for most NRTIs. Consequently, mitochondria are particularly susceptible to inhibition of DNA replication by the NRTIs, which is consistent with the phenotype of mitochondrial depletion and metabolic failure in affected patients. However, the DNA pol-gamma hypothesis by itself fails to explain the entire array of metabolic deficiencies associated with NRTI-induced disorders. In this article, we review the published literature regarding the direct effects of NRTIs on various mitochondrial targets and suggest the possibility that the initiating event in NRTI-induced cardiomyopathy is a direct mitochondrial toxicity rather than inhibition of mitochondrial DNA pol-gamma. The goal of this review is to encourage a discussion of the cause of NRTI-induced mitochondrial cardiomyopathy to include a fresh consideration of all possible targets and integrating pathways that are involved in establishing mitochondrial bioenergetic fidelity and metabolic capacity in the affected myocardium.

Direct effects of nucleoside reverse transcriptase inhibitors on rat cardiac mitochondrial bioenergetics.

In this investigation we demonstrate that various nucleoside reverse transcriptase inhibitors (NRTIs) and their corresponding nucleotides can cause a direct, DNA polymerase-gamma-independent, inhibition of respiration, membrane potential, and calcium loading capacity in isolated rat heart mitochondria in vitro. Both AZT and d4T also increased total adenine phosphate energy charge in H9c2 rat cardiac myocytes in cell culture. These results demonstrate that the various NRTI nucleosides and nucleotides are capable, at sufficiently high concentrations, of directly affecting mitochondrial bioenergetics in vitro, which may enhance the toxicity observed in vivo previously attributed to inhibition of DNA polymerase-gamma.