Simultaneous characterization of multiple Psoraleae Fructus bioactive compounds in rat plasma by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry for application in sex-related differences in pharmacokinetics.

A method for the simultaneous quantification of 13 bioactive compounds (psoralen, isopsoralen, isobavachin, bakuchalcone, neobabaisoflavone, bavachin, corylin, psoralidin, isobavachalcone, bavachinin, corylifol A, bavachalcone, and bakuchiol) by ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry has been developed and validated in rat plasma. Osthol was used as an internal standard and plasma samples were pretreated with one-step liquid-liquid extraction. These analytes were separated using a gradient mobile phase system of water and acetonitrile at a flow rate of 0.2 mL/min on a reverse-phase C18 column and analyzed in the selected multiple reactions monitoring mode. All calibration curves were linear (r > 0.9952) over the tested ranges. The intra- and interday accuracy and precisions of these analytes at three different concentration levels were within the acceptable limits of <15% at all concentrations. The mean recoveries of these analytes at three concentrations were more than 60.2% and the matrix effects were in the range of 85-115%. Stability studies proved that the analytes were stable under the tested conditions. The developed method was applied to evaluating the pharmacokinetic study of 13 bioactive compounds after oral administration of Psoraleae Fructus in rat of different genders. Some active compounds in Psoraleae Fructus had sex-related pharmacokinetics.

Never-in-mitosis A-related kinase 8, a novel target of von-Hippel-Lindau tumor suppressor protein, promotes gastric cancer cell proliferation.

Previous research has revealed that the von-Hippel-Lindau tumor suppressor protein (pVHL) may downregulate never-in-mitosis A-related kinase 8 (NEK8) via hypoxia-inducible factor-α (HIF-α). The HIF-independent functions of pVHL also serve an important role in its tumor-suppressor action. In the present study, the association between pVHL and NEK8 was demonstrated in the human gastric cancer cell line, SGC-7901, indicating a direct interaction of pVHL with NEK8. Subsequently, it was reported that MG-132, a specific proteasome inhibitor, may attenuate pVHL overexpression-induced reductions in NEK8 protein expression levels. In addition, the present study revealed that pVHL may stimulate the rapid degradation of NEK8 protein and promote its ubiquitination. The association between the expression profile of NEK8 and the survival status of patients with gastric cancer was analyzed from an online database. Kaplan-Meier survival plots indicated that higher expression levels of NEK8 may lead to poor survival, as suggested by the transcriptomic data of 1,065 patients with gastric cancer. It was found that NEK8-knockdown mediated by RNA interference inhibited SGC-7901 and SNU-1 proliferation, colony formation and migration in vitro, and tumor growth in vivo. Collectively, the present study proposed that NEK8 may be a novel target of pVHL as a ubiquitin E3 ligase, and may serve a role as a potential oncoprotein in human gastric cancer.

VHL loss predicts response to Aurora kinase A inhibitor in renal cell carcinoma cells.

The majority of molecular targets of anticancer agents are limited to a subset of patients, and therefore identification of more specific biomarkers that can be used to improve clinical outcomes is of increasing interest. The present study showed that von Hippel­Lindau tumor suppressor (VHL) tumor­suppressor activity may influence the therapeutic response to Aurora kinase A (AURKA) inhibitors in human renal cell carcinoma (RCC). VHL protein (pVHL) expression was evaluated by immunoblotting in the human RCC cell lines CAKI, ACHN, 786­O, 769­P and A498. The anti­tumor activities of alisertib, an AURKA­specific chemical inhibitor, were detected by Cell Counting Kit­8 assay in vitro and mouse xenograft model in vivo. Additionally, the VHL­mediated anti­tumor activity was assessed in 769­P and CAKI cells via the loss or gain of VHL. The results revealed that VHL­deficient 786­O, 769­P and A498 cells were sensitive to alisertib. By contrast, alisertib­resistant CAKI and ACHN cells expressed the wild type VHL gene. In addition, rescue or knockdown of VHL was observed to increase or decrease alisertib anti­proliferation activity, respectively, in RCC cells. The inverse correlation between the VHL gene expression profile and alisertib sensitivity was further confirmed in human cancer xenografts models. Taken together, these results suggested that VHL loss could potentially serve as a biomarker for predicting the efficacy of AURKA inhibitors.

COH-203, a novel microtubule inhibitor, exhibits potent anti-tumor activity via p53-dependent senescence in hepatocellular carcinoma.

5-(3-Hydroxy-4-methoxyphenyl)-4-(3,4,5-trimethoxyphenyl)-3H-1,2-dithiol-3-one (COH-203) is a novel synthesized analogue of combretastatin A-4 that can be classified as a microtubule inhibitor. In this study, we evaluated the anti-hepatoma effect of COH-203 in vitro and in vivo and explored the underlying molecular mechanisms. COH-203 was shown to be more effective in inhibiting the proliferation of liver cancer cells compared with normal liver cells. COH-203 also displayed potent anti-tumor activity in a hepatocellular carcinoma xenograft model without significant toxicity. Mechanistic studies demonstrated that treatment with COH-203 induced mitotic arrest by inhibiting tubulin polymerization in BEL-7402 liver cancer cells. Long-term COH-203 treatment in BEL-7402 cells led to mitotic slippage followed by senescence via the p14(Arf)-p53-p21 and p16(INK4α)-Rb pathways. Furthermore, suppression of p53 via pifithrin-α (p53 inhibitor) and p53-siRNA attenuated COH-203-induced senescence in BEL-7402 cells, suggesting that COH-203 induced senescence p53-dependently. In conclusion, we report for the first time that COH-203, one compound in the combretastatin family, promotes anti-proliferative activity through the induction of p-53 dependent senescence. Our findings will provide a molecular rationale for the development of COH-203 as a promising anti-tumor agent.

Exogenous γ-aminobutyric acid (GABA) affects pollen tube growth via modulating putative Ca2+-permeable membrane channels and is coupled to negative regulation on glutamate decarboxylase.

γ-Aminobutyric acid (GABA) is implicated in pollen tube growth, but the molecular and cellular mechanisms that it mediates are largely unknown. Here, it is shown that exogenous GABA modulates putative Ca(2+)-permeable channels on the plasma membranes of tobacco pollen grains and pollen tubes. Whole-cell voltage-clamp experiments and non-invasive micromeasurement technology (NMT) revealed that the influx of Ca(2+) increases in pollen tubes in response to exogenous GABA. It is also demonstrated that glutamate decarboxylase (GAD), the rate-limiting enzyme of GABA biosynthesis, is involved in feedback controls of Ca(2+)-permeable channels to fluctuate intracellular GABA levels and thus modulate pollen tube growth. The findings suggest that GAD activity linked with Ca(2+)-permeable channels relays an extracellular GABA signal and integrates multiple signal pathways to modulate tobacco pollen tube growth. Thus, the data explain how GABA mediates the communication between the style and the growing pollen tubes.

Selective inhibition of CCR7(-) effector memory T cell activation by a novel peptide targeting Kv1.3 channel in a rat experimental autoimmune encephalomyelitis model.

The voltage-gated Kv1.3 K(+) channel in effector memory T cells serves as a new therapeutic target for multiple sclerosis. In our previous studies, the novel peptide ADWX-1 was designed and synthesized as a specific Kv1.3 blocker. However, it is unclear if and how ADWX-1 alleviates experimental autoimmune encephalomyelitis, a model for multiple sclerosis. In this study, the administration of ADWX-1 significantly ameliorated the rat experimental autoimmune encephalomyelitis model by selectively inhibiting CD4(+)CCR7(-) phenotype effector memory T cell activation. In contrast, the Kv1.3-specific peptide had little effect on CD4(+)CCR7(+) cells, thereby limiting side effects. Furthermore, we determined that ADWX-1 is involved in the regulation of NF-κB signaling through upstream protein kinase C-θ (PKCθ) in the IL-2 pathway of CD4(+)CCR7(-) cells. The elevated expression of Kv1.3 mRNA and protein in activated CD4(+)CCR7(-) cells was reduced by ADWX-1 engagement; however, an apparent alteration in CD4(+)CCR7(+) cells was not observed. Moreover, the selective regulation of the Kv1.3 channel gene expression pattern by ADWX-1 provided a further and sustained inhibition of the CD4(+)CCR7(-) phenotype, which depends on the activity of Kv1.3 to modulate its activation signal. In addition, ADWX-1 mediated the activation of differentiated Th17 cells through the CCR7(-) phenotype. The efficacy of ADWX-1 is supported by multiple functions, which are based on a Kv1.3(high) CD4(+)CCR7(-) T cell selectivity through two different pathways, including the classic channel activity-associated IL-2 pathway and the new Kv1.3 channel gene expression pathway.

Structural and functional diversity of acidic scorpion potassium channel toxins.

BACKGROUND: Although the basic scorpion K(+) channel toxins (KTxs) are well-known pharmacological tools and potential drug candidates, characterization the acidic KTxs still has the great significance for their potential selectivity towards different K(+) channel subtypes. Unfortunately, research on the acidic KTxs has been ignored for several years and progressed slowly. PRINCIPAL FINDINGS: Here, we describe the identification of nine new acidic KTxs by cDNA cloning and bioinformatic analyses. Seven of these toxins belong to three new α-KTx subfamilies (α-KTx28, α-KTx29, and α-KTx30), and two are new members of the known κ-KTx2 subfamily. ImKTx104 containing three disulfide bridges, the first member of the α-KTx28 subfamily, has a low sequence homology with other known KTxs, and its NMR structure suggests ImKTx104 adopts a modified cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif that has no apparent α-helixs and ß-sheets, but still stabilized by three disulfide bridges. These newly described acidic KTxs exhibit differential pharmacological effects on potassium channels. Acidic scorpion toxin ImKTx104 was the first peptide inhibitor found to affect KCNQ1 channel, which is insensitive to the basic KTxs and is strongly associated with human cardiac abnormalities. ImKTx104 selectively inhibited KCNQ1 channel with a K(d) of 11.69 µM, but was less effective against the basic KTxs-sensitive potassium channels. In addition to the ImKTx104 toxin, HeTx204 peptide, containing a cystine-stabilized α-helix-loop-helix (CS-α/α) fold scaffold motif, blocked both Kv1.3 and KCNQ1 channels. StKTx23 toxin, with a cystine-stabilized α-helix-loop-ß-sheet (CS-α/ß) fold motif, could inhibit Kv1.3 channel, but not the KCNQ1 channel. CONCLUSIONS/SIGNIFICANCE: These findings characterize the structural and functional diversity of acidic KTxs, and could accelerate the development and clinical use of acidic KTxs as pharmacological tools and potential drugs.

Hg1, novel peptide inhibitor specific for Kv1.3 channels from first scorpion Kunitz-type potassium channel toxin family.

The potassium channel Kv1.3 is an attractive pharmacological target for autoimmune diseases. Specific peptide inhibitors are key prospects for diagnosing and treating these diseases. Here, we identified the first scorpion Kunitz-type potassium channel toxin family with three groups and seven members. In addition to their function as trypsin inhibitors with dissociation constants of 140 nM for recombinant LmKTT-1a, 160 nM for LmKTT-1b, 124 nM for LmKTT-1c, 136 nM for BmKTT-1, 420 nM for BmKTT-2, 760 nM for BmKTT-3, and 107 nM for Hg1, all seven recombinant scorpion Kunitz-type toxins could block the Kv1.3 channel. Electrophysiological experiments showed that six of seven scorpion toxins inhibited ~50-80% of Kv1.3 channel currents at a concentration of 1 µM. The exception was rBmKTT-3, which had weak activity. The IC(50) values of rBmKTT-1, rBmKTT-2, and rHg1 for Kv1.3 channels were ~129.7, 371.3, and 6.2 nM, respectively. Further pharmacological experiments indicated that rHg1 was a highly selective Kv1.3 channel inhibitor with weak affinity for other potassium channels. Different from classical Kunitz-type potassium channel toxins with N-terminal regions as the channel-interacting interfaces, the channel-interacting interface of Hg1 was in the C-terminal region. In conclusion, these findings describe the first scorpion Kunitz-type potassium channel toxin family, of which a novel inhibitor, Hg1, is specific for Kv1.3 channels. Their structural and functional diversity strongly suggest that Kunitz-type toxins are a new source to screen and design potential peptides for diagnosing and treating Kv1.3-mediated autoimmune diseases.

Reactive oxygen species contribute to oridonin-induced apoptosis and autophagy in human cervical carcinoma HeLa cells.

AIM: To investigate the role of reactive oxygen species (ROS) in oridonin-induced apoptosis and autophagy in HeLa cells. METHODS: The cell viability was measured using MTT assay. Morphological changes of apoptosis and autophagy were examined using Hoechst 33258 staining and monodansylcadaverine (MDC) staining, respectively. The mitochondrial membrane potential (ΔΨm) was measured using fluorescent dye rhodamine 123. DCF-induced fluorescence was used to measure the intracellular ROS level. Protein expression was examined using Western blot. RESULTS: Treatment of HeLa cells with oridonin (20-160 µmol/L) inhibited the cell growth in time- and concentration-dependent manners. The cells treated with oridonin (80 µmol/L) for 24 h displayed marked DNA fragmentation and MDC-positive autophagosomes. In the presence of the specific autophagy inhibitor 3-MA (2 mmol/L), the oridonin-induced apoptosis was significantly enhanced. Treatment of HeLa cells with oridonin (20-120 µmol/L) induced intracellular ROS generation in a concentration-dependent manner. In the presence of the ROS scavenger NAC (5 mmol/L), the oridinin-induced ROS generation was markedly reduced. NAC (5 mmol/L) or non-thiol antioxidant catalase (1000 U/mL) significantly reduced the oridonin-induced inhibition of cell growth and apoptosis. Furthermore, oridonin significantly reduced ΔΨm, which was blocked by NAC. Oridonin markedly increased Bax expression in mitochondria, and decreased Bcl-2 expression in both the cytosol and mitochondria. Oridonin also markedly increased the phosphorylation of Bcl-2 in the cytosol. All the effects were blocked by NAC. Oridonin increased the levels of caspase-3 and caspase-8, and decreased the expression of pro-caspase 3 and pro-caspase 9, which were blocked by NAC. CONCLUSION: ROS plays a critical role in oridonin-induced apoptosis and autophagy.

Effects of acute and chronic administration of MK-801 on c-Fos protein expression in mice brain regions implicated in schizophrenia with or without clozapine.

This study investigated the effects of acute and chronic administration of the non-competitive NMDA receptor antagonists MK-801 on c-Fos protein expression in different brain regions of mice with or without clozapine. MK-801 (0.6 mg/kg) acute administration produced a significant increase in the expression of c-Fos protein in the layers III-IV of posterior cingulate and retrosplenial (PC/RS) cortex, which was consistent with the previous reports. Moreover, we presented a new finding that MK-801 (0.6 mg/kg) chronic administration for 8 days produced a significant increase of c-Fos protein expression in the PC/RS cortex, prefrontal cortex (PFC) and hypothalamus of mice. Among that, c-Fos protein expression in the PC/RS cortex of mice was most significant. Compared to acute administration, we found that MK-801 chronic administration significantly increased the expression of c-Fos protein in the PC/RS cortex, PFC and hypothalamus. Furthermore, pretreatment of mice with clozapine significantly decreased the expression of c-Fos protein induced by MK-801 acute and chronic administration. These results suggest that c-Fos protein, the marker of neuronal activation, might play an important role in the chronic pathophysiological process of schizophrenic model induced by NMDA receptor antagonist.

[Apoptosis induced by NNAMB, a novel polyamine conjugate, in human erythroleukemia K562 cells and its mechanism].

OBJECTIVE: To investigate the apoptosis-inducing effects of NNAMB, a novel polyamine conjugate, in erythroleukemia K562 cells and its molecular mechanism. METHODS: Cell viability was assessed by MTT assay and trypan blue dye exclusion method. The cell morphology was observed by fluorescence microscopy. The cell cycle distribution, apoptosis and mitochondrial membrane potential were measured by flow cytometry. The expression of caspase-3, -8, -9, cytochrome c in the K562 cells was detected by Western blot. RESULTS: NNAMB inhibited the proliferation of K562 cells. The cells treated with NNAMB showed a typical apoptotic morphology, Sub-G1 peak and loss of mitochondrial membrane potential. Western blot assay showed that NNAMB increased the expression of caspase-3, -9, cytochrome c but not caspase-8 in a dose-and time-dependent manner. CONCLUSION: NNAMB induces apoptosis via mitochondrial pathway in K562 cells.

Structural basis of a potent peptide inhibitor designed for Kv1.3 channel, a therapeutic target of autoimmune disease.

The potassium channel Kv1.3 is an attractive pharmacological target for immunomodulation of T cell-mediated autoimmune diseases. Potent and selective blockers of Kv1.3 are potential therapeutics for treating these diseases. Here we describe the design of a new peptide inhibitor that is potent and selective for Kv1.3. Three residues (Gly(11), Ile(28), and Asp(33)) of a scorpion toxin BmKTX were substituted by Arg(11), Thr(28), and His(33), resulting in a new peptide, named ADWX-1. The ADWX-1 peptide blocked Kv1.3 with picomolar affinity (IC(50), 1.89 pM), showing a 100-fold increase in activity compared with the native BmKTX toxin. The ADWX-1 also displayed good selectivity on Kv1.3 over related Kv1.1 and Kv1.2 channels. Furthermore, alanine-scanning mutagenesis was carried out to map the functional residues of ADWX-1 in blocking Kv1.3. Moreover, computational simulation was used to build a structural model of the ADWX-1-Kv1.3 complex. This model suggests that all mutated residues are favorable for both the high potency and selectivity of ADWX-1 toward Kv1.3. While Arg(11) of ADWX-1 interacts with Asp(386) in Kv1.3, Thr(28) and His(33) of ADWX-1 locate right above the selectivity filter-S6 linker of Kv1.3. Together, our data indicate that the specific ADWX-1 peptide would be a viable lead in the therapy of T cell-mediated autoimmune diseases, and the successful design of ADWX-1 suggests that rational design based on the structural model of the peptide-channel complex should accelerate the development of diagnostic and therapeutic agents for human channelopathies.

Synergistic antitumor effects of anthracenylmethyl homospermidine and alpha-difluoromethylornithine on promyelocytic leukemia HL60 cells.

The present study was designed to assess the synergistic antitumor effects of anthracenylmethyl homospermidine (ANTMHspd), a novel polyamine conjugate, with alpha-difluoromethylornithine (DFMO) and to elucidate the mechanism of these effects on human leukemia HL60 cells. Cell proliferation was assessed by the MTT assay. Cell cycle, apoptosis and mitochondria membrane potential (MMP) were evaluated by flow cytometry. Caspases and cytochrome c were detected by Western Blot analysis. The combination treatment strongly inhibited cell proliferation, induced cell apoptosis and caused an accumulation in the G1 phase with an accompaniment decrease in S phase. Moreover, reduction of MMP, release of cytochrome c and activation of caspase-3 and caspase-9 but not caspase-8 were observed during the combination-mediated apoptosis. All these findings demonstrated that the combination treatment with DFMO and ANTMHspd resulted in synergistic antitumor effects on HL60 cells.

Molecular cloning and electrophysiological studies on the first K(+) channel toxin (LmKTx8) derived from scorpion Lychas mucronatus.

LmKTx8, the first toxic gene isolated from the venom of scorpion Lychas mucronatus by constructing cDNA library method, was expressed and characterized physiologically. The mature peptide has 40 residues including six conserved cysteines, and is classified as one of alpha-KTx11 subfamily. Using patch-clamp recording, the recombinant LmKTx8 (rLmKTx8) was used to test the effect on voltage-gated K(+) channels (Kv1.3) stably expressed in COS7 cells and large conductance-Ca(2+)-activated K(+) (BK) channels expressed in HEK293. The results of electrophysiological experiments showed that the rLmKTx8 was a potent inhibitor of Kv1.3 channels with an IC(50)=26.40+/-1.62nM, but 100nM rLmKTx8 did not block the BK currents. LmKTx8 or its analogs might serve as a potential candidate for the development of new drugs for autoimmune diseases.

A novel homospermidine conjugate inhibits growth and induces apoptosis in human hepatoma cells.

AIM: To elucidate the mechanism responsible for the antiproliferative effects of a novel homospermidine conjugate, anthracenylmethyl homospermidine (ANTMHspd), in the human hepatoma BEL-7402 cell line. METHODS: The viability of the cells was assessed by MTT assay and the trypan blue dye exclusion method. Morphological changes were observed by fluorescence microscopy with Hoechst 33258 staining. Cell cycle distribution, apoptosis, and mitochondrial membrane potential were measured by flow cytometry. Protein expression was detected by Western blot analysis. RESULTS: ANTMHspd strongly decreased BEL-7402 cell proliferation in a dose- and time-dependent manner. Hoechst 33258 staining and the flow cytometry assay showed that ANTMHspd induced cell apoptosis and cell cycle perturbation. Furthermore, ANTMHspd could induce mitochondrial membrane potential loss and cytochrome c release and enhance cleaved caspase-3, cleaved caspase-9, and Bax protein expression without caspase-8 activation. ANTMHspd could also decrease the expression of Bcl-2 and cytochrome c in mitochondria. In addition, the specific inhibitors of caspase-9 and caspase-3 almost abolished the ANTMHspd-induced caspase-9 and caspase-3 activation, respectively. CONCLUSION: ANTMHspd could induce BEL-7402 cell apoptosis via the mitochondrial/caspase-dependent pathway and the Bcl-2 family was involved in the control of apoptosis.

Clonidine, moxonidine, folic acid, and mecobalamin improve baroreflex function in stroke-prone, spontaneously hypertensive rats.

AIM: To investigate the effect of clonidine, moxonidine, folic acid, and mecobalamin on arterial baroreflex (ABR) function in stroke-prone spontaneously hypertensive rats (SHR-SP) and the possible mechanisms involved. METHODS: Eighty-one SHR-SP were divided into 7 groups. Four groups were designated for the intragastric (ig) administration of clonidine (1.0 and 10.0 microg/kg), moxonidine (0.1 and 1.0 mg/kg), folic acid (1.0 mg/kg), and mecobalamin (1.0 mg/kg). Three groups were for the intracerebroventricular (icv) injection of clonidine (4 microg/4 microL), moxonidine (5 microg/4 microL), and mecobalamin (20 microg/4 microL). Blood pressure (BP) was recorded in the conscious state for 30 min and baroreflex sensitivity (BRS) was determined respectively before and after drug administration. RESULTS: Clonidine and moxonidine significantly decreased BP, prolonged the heart period (HP), and increased BRS when administered as either ig or icv injections. Both BP and HP were unchanged by ig folic acid or mecobalamin injection. However, BRS was significantly increased by both. CONCLUSION: Clonidine, moxonidine, folic acid, and mecobalamin improved impaired ABR function in SHR-SP. The central mechanism was involved in this effect of either clonidine or moxonidine. Mecobalamin improved ABR function through the peripheral mechanism.

Synthesis and anti-proliferative in-vitro activity of two natural dihydrostilbenes and their analogues.

A total synthetic route for two natural dihydrostilbenes with significant cytotoxicity toward human cancer cell lines, (3-(2-(7-methoxybenzo[d][1,3]dioxol-5-yl)ethyl)phenol 1a and 6-(3-hydroxyphenethyl)benzo[d][1,3]dioxol-4-ol 1b), which were isolated from Bulbophyllum odoratissimum Lindl, was developed via Wittig-Horner reaction. The natural products 1a and 1b were obtained in 28% and 20% overall yield, respectively. Additionally, nine analogues, 1c-1k, of the two natural dihydrostilbenes were synthesized and evaluated for their anti-proliferative activity against human SGC-7901, KB and HT-1080 cell lines by MTT assay. The activities of 1c and 1d were in the same range as those of the natural products 1a and 1b.

Involvement of mitochondria and caspase pathways in N-demethyl-clarithromycin-induced apoptosis in human cervical cancer HeLa cell.

AIM: To study the mechanisms by which N-demethyl-clarithromycin (NDC) induces human cervical cancer HeLa cell apoptosis in vitro. METHODS: The viability of N-demethyl-clarithromycin-induced HeLa cells was measured by MTT assay. Apoptotic cells with condensed nuclei were visualized by phase contrast microscopy. Nucleosomal DNA fragmentation was assayed by agarose gel electrophoresis. Measurement of mitochondrial transmembrane potential was analyzed by a FACScan flowcytometer. Caspase-3, poly-(ADP-ribose) polymerase (PARP), caspase-activated DNase (ICAD), Bcl-2, Bax, p53, and SIRT1 protein expression and the release of cytochrome c were detected by Western blot analysis. RESULTS: N-demethyl-clarithromycin, an anti-inflammatory substance, inhibited HeLa cell growth in a dose- and time-dependent manner. N-demethyl-clarithro-mycin induced HeLa cell death through the apoptotic pathways. The pan-caspase inhibitor (z-VAD-fmk), caspase-3 inhibitor (z-DEVD-fmk) and the caspase-9 inhibitor (z-LEHD-fmk) partially enhanced cell viability induced by N-demethyl-clarithromycin, but the caspase-8 inhibitor (z-IETD-fmk) had almost no effect. Caspase-3 was activated then followed by the degradation of caspase-3 substrates, the inhibitor of ICAD and PARP. Simultaneously, mitochondrial transmembrane potential was markedly reduced and the release of cytochrome c in the cytosol was increased. N-demethyl-clarithromycin upregulated the expression ratio of mitochondrial Bax/Bcl-2, and significantly increased the expression of the p53 protein. It also downregulated anti-apoptotic protein SIRT1 expression. CONCLUSION: N-demethyl-clarithromycin induced apoptosis in HeLa cells via the mitochondrial pathway.