Apigeninidin induces apoptosis through activation of Bak and Bax and subsequent mediation of mitochondrial damage in human promyelocytic leukemia HL-60 cells

Treatment of human promyelocytic leukemia HL-60 cells with apigeninidin could induce cytotoxicity (IC₅₀ = ～80 μM), along with apoptotic sub-G₁ cells, TUNEL-positive apoptotic DNA fragmentation, activation of the multidomain pro-apoptotic Bcl-2 proteins (Bak and Bax), mitochondrial membrane potential (Δψ_m) loss, release of mitochondrial cytochrome c and AIF into the cytoplasm, activation of caspase-9, -3, -8, and -7, and cleavage of PARP and lamin B. These induced apoptotic events were accompanied by decrease of Bcl-2 level and increase of Bak and Bax levels. Apigeninidin-induced sub-G₁ cells and activation of Bak and Bax were also detected in human acute leukemia Jurkat T cells, but not in Jurkat T cells overexpressing Bcl-2. Pretreatment of HL-60 cells with the pan-caspase inhibitor z-VAD-fmk reduced significantly apigeninidin-induced sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak and Bax activations, Δψ_m loss, and release of mitochondrial cytochrome c and AIF. None of FADD and caspase-8 deficiencies affected the sensitivity of Jurkat T cells to apigeninidin-induced cytotoxicity. These results demonstrated that apigeninidin-induced apoptosis was mediated by activation of Bak and Bax, mitochondrial damage and resultant release of not only cytochrome c, causing caspase cascade activation, but also caspase-independent death effector AIF in HL-60 cells.     

CWJ-081, a novel 3-arylisoquinoline derivative,induces apoptosis in human leukemia HL-60 cells partially involves reactive oxygen species through c-Jun NH2-terminal kinase pathway

In the present study, we investigated the effect of a novel 3-arylisoquinoline derivative 3-(6-ethyl-benzo[1,3]dioxol-5-yl)-7,8-dimethoxy-2-methyl-2H-isoquinolin-1-one (CWJ-081) on the induction of apoptosis and the putative molecular mechanism of its action in human leukemia cells. Treatment with CWJ-081 exhibited a characteristic feature of apoptosis including externalization of phosphatidylserine and formation of DNA fragmentation in human leukemia cell lines (HL-60, U-937, K-562). In addition, stimulation of HL-60 cells with CWJ-081 induced a series of intracellular events: (1) the activations of caspase-8, -9, and -3; (2) the cleavage of poly (ADP-ribose) polymerase-1 (PARP-1); (3) the loss of mitochondrial membrane potential (ΔΨ_m); (4) the release of cytochrome c; and (5) the modulation of Bcl-2 family proteins. We further demonstrated that CWJ-081 induces reactive oxygen species (ROS) production and c-Jun NH₂-terminal kinase (JNK) activation. Pretreatment with the antioxidant N-acetyl-l-cysteine (NAC) markedly inhibited the CWJ-081-induced JNK activation and apoptosis. Moreover, CWJ-081-induced apoptosis was suppressed in the presence of SP600125, a specific JNK inhibitor. Taken together, these data suggest that CWJ-081 induces apoptosis via the mitochondrial apoptotic pathway in HL-60 cells, and ROS-mediated JNK activation plays a key role in the CWJ-081-induced apoptosis.     

Antimycin A-induced killing of HL-60 cells: apoptosis initiated from within mitochondria does not necessarily proceed via caspase 9.

BACKGROUND: Antimycin A (AMA) inhibits mitochondrial electron transport, collapses the mitochondrial membrane potential, and causes the production of reactive oxygen species. Previous work by me and my colleagues has demonstrated that AMA causes an array of typical apoptotic phenomena in HL-60 cells. The hypothesis that AMA causes HL-60 apoptosis by the intrinsic apoptotic pathway has now been tested. METHODS: Z-LEHD-FMK and Z-IETD-FMK were used as specific inhibitors of the initiator caspases 9 and 8, respectively. Caspase 3 activation, DNA fragmentation, and cellular disintegration were measured by flow cytometry. Cytochrome c release, chromatin condensation, and nuclear fragmentation were measured by microscopy. RESULTS: AMA caused mitochondrial cytochrome c release and neither Z-LEHD-FMK nor Z-IETD-FMK inhibited that. In the absence of caspase inhibition there was a very close correlation between cytochrome c release and caspase 3 activation. Z-LEHD-FMK blocked caspase 3 activation but enhanced DNA fragmentation and failed to stop nuclear or cellular disintegration. Z-IETD-FMK also blocked caspase 3 activation but, in contrast to Z-LEHD-FMK, delayed DNA fragmentation and disintegration of the nucleus and the cell. CONCLUSIONS: The hypothesis to explain AMA-induced HL-60 apoptosis was clearly inadequate because: (a) caspase 9 inhibition did not prevent DNA fragmentation or cell death, (b) apoptosis proceeded in the absence of caspase-3 activation, (c) the main pathway leading to activation of the executioner caspases was by caspase-8 activation, but caspase 8 inhibition only delayed apoptosis, and (d) activation of caspases 8 and 9 may be necessary for caspase-3 activation. Thus, in this cell model, apoptosis triggered from within the mitochondria does not necessarily proceed by caspase 9, and caspase 3 is not critical to apoptosis. The results provide further evidence that, when parts of the apoptotic network are blocked, a cell is able to complete the program of cell death by alternate pathways.     

Capillin,a major constituent of Artemisia capillaris Thunb. flower essential oil,induces apoptosis through the mitochondrial pathway in human leukemia HL-60 cells

BackgroundNatural products are one of the most important sources of drugs used in pharmaceutical therapeutics. Screening of several natural products in the search for novel anticancer agents against human leukemia HL-60 cells led us to identify potent apoptosis-inducing activity in the essential oil fraction from Artemisia capillaris Thunb. flower.MethodsThe cytotoxic effects of extracts were assessed on human leukemia HL-60 cells by XTT assay. Induction of apoptosis was assessed by analysis of DNA fragmentation and nuclear morphological change. The plant name was checked with the plant list website (http://www.theplantlist.org).ResultsA purified compound from the essential oil fraction from Artemisia capillaris Thunb. flower that potently inhibited cell growth in human leukemia HL-60 cells was identified as capillin. The cytotoxic effect of capillin in cells was associated with apoptosis. When HL-60 cells were treated with 10⁻⁶ M capillin for 6 h, characteristic features of apoptosis such as DNA fragmentation and nuclear fragmentation were observed. Moreover, activation of c-Jun N-terminal kinase (JNK) was detected after treatment with capillin preceding the appearance of characteristic properties of apoptosis. Release of cytochrome c from mitochondria was also observed in HL-60 cells that had been treated with capillin.ConclusionCapillin induces apoptosis in HL-60 cells via the mitochondrial apoptotic pathway, which might be controlled through JNK signaling. Our results indicate that capillin may be a potentially useful anticancer drug that could enhance therapeutic efficacy.     

In vitro anticancer activity of loquat tea by inducing apoptosis in human leukemia cells

Fresh loquat leaves have been used as folk health herb in Asian countries for long time, although the evidence supporting their functions is still minimal. This study aimed to clarify the chemopreventive effect of loquat tea extract (LTE) by investigating the inhibition on proliferation, and underlying mechanisms in human promyelocytic leukemia cells (HL-60). LTE inhibited proliferation of HL-60 in a dose-dependent manner. Molecular data showed that the isolated fraction of LTE induced apoptosis of HL-60 as characterized by DNA fragmentation; activation of caspase-3, -8, and -9; and inactivation of poly(ADP)ribose polymerase. Moreover, LTE fraction increased the ratio of pro-apoptotic Bcl-2-associated X protein (Bax)/anti-apoptotic myeloid cell leukemia 1 (Mcl-1) that caused mitochondrial membrane potential loss and cytochrome c released to cytosol. Thus, our data indicate that LTE might induce apoptosis in HL-60 cells through a mitochondrial dysfunction pathway. These findings enhance our understanding for chemopreventive function of loquat tea.     

Resveratrol induces mitochondria-mediated AIF and to a lesser extent caspase-9-dependent apoptosis in human lung adenocarcinoma ASTC-a-1 cells

Resveratrol (RV), a natural plant polyphenol widely present in foods such as grapes, wine, and peanuts, has an ability to inhibit various stages of carcinogenesis in vitro and in vivo. In this report, we explored the roles of intrinsic and extrinsic apoptotic pathways during RV-induced apoptosis in human lung adenocarcinoma (ASTC-a-1) cells. After exposure of cells to different concentrations of RV, we found that RV induced concentration-dependent apoptosis. Fluorometric substrates assay and western blotting (WB) analysis showed that caspase-8 was not activated, which was further verified by monitoring the cleavage of Bid to tBid using fluorescence resonance energy transfer (FRET) microscopy imaging inside single living cells, indicating that extrinsic apoptotic pathway was not involved in RV-induced apoptosis. In addition, inhibition of caspases-3 or -9 but not caspase-8 using the specific inhibitors of caspases modestly but significantly attenuated RV-induced apoptosis. Moreover, flow cytometry (FCM) analysis showed that RV treatment induced time-dependent loss of mitochondrial membrane potential (?ψ(m)), in combination with the activation of caspases-3 and -9; we therefore concluded that RV-induced apoptosis involved the intrinsic apoptotic pathway. It is noteworthy that RV treatment induced translocation of AIF from mitochondria to nucleus in a time dependent manner, and that knockdown of AIF remarkably attenuated RV-induced apoptosis. Collectively, our findings demonstrate that RV induces caspase-8-independent apoptosis via AIF and to a lesser extent caspase-9-dependent mitochondrial pathway in ASTC-a-1 cells.     

Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.     

Down-regulation of procaspase-8 expression by focal adhesion kinase protects HL-60 cells from TRAIL-induced apoptosis

We have demonstrated that focal adhesion kinase (FAK)-overexpressed (HL-60/FAK) cells have marked resistance against various apoptotic stimuli such as hydrogen peroxide, etoposide, and ionizing radiation compared with the vector-transfected (HL-60/Vect) cells. HL-60/FAK cells are highly resistant to TRAIL-induced apoptosis, while original HL-60 or HL-60/Vect cells were sensitive. TRAIL at 500 ng/ml induced significant DNA fragmentation, activation of caspase-8 and 3, the processing of a proapoptotic BID, and mitochondrial release of cytochrome c in HL-60/Vect cells, whereas no such events were observed in the HL-60/FAK cells. In particular, the expression of procaspase-8 gene and subsequent cleavage of caspase-8 were markedly reduced in HL-60/FAK cells, while expression of TRAIL-receptor 2 and 3, TRADD, and FADD was equivalent in both types of cells. In HL-60/FAK cells, the phosphoinositide 3 (PI3)-kinase/Akt survival pathway was constitutively activated, accompanied by significant induction of inhibitor-of-apoptosis proteins, XIAP, RIP, and Bcl-XL. The introduction of FAK siRNA in HL-60/FAK cells sensitized them against TRAIL-induced apoptosis, confirming that overexpressed FAK downregulates procaspase-8 expression, which subsequently inhibits downstream apoptosis pathway in the HL-60/FAK cells.     

Reactive oxygen species generation and mitochondrial dysfunction in the apoptotic cell death of human myeloid leukemia HL-60 cells by a dietary compound withaferin A with concomitant protection by <Emphasis Type="Italic">N</Emphasis>-acetyl cysteine

Induction of apoptosis in cancer cells has become the major focus of anti-cancer therapeutics development. WithaferinA, a major chemical constituent of Withania somnifera, reportedly shows cytotoxicity in a variety of tumor cell lines while its molecular mechanisms of action are not fully understood. We observed that withaferinA primarily induces oxidative stress in human leukemia HL-60 cells and in several other cancer cell lines. The withanolide induced early ROS generation and mitochondrial membrane potential (Δψ_mt) loss, which preceded release of cytochrome c, translocation of Bax to mitochondria and apoptosis inducing factor to cell nuclei. These events paralleled activation of caspases −9, −3 and PARP cleavage. WA also activated extrinsic pathway significantly as evidenced by time dependent increase in caspase-8 activity vis-à-vis TNFR-1 over expression. WA mediated decreased expression of Bid may be an important event for cross talk between intrinsic and extrinsic signaling. Furthermore, withaferinA inhibited DNA binding of NF-κB and caused nuclear cleavage of p65/Rel by activated caspase-3. N-acetyl-cysteine rescued all these events suggesting thereby a pro-oxidant effect of withaferinA. The results of our studies demonstrate that withaferinA induced early ROS generation and mitochondrial dysfunction in cancer cells trigger events responsible for mitochondrial -dependent and -independent apoptosis pathways.     

Induction of apoptosis through caspase-independent or caspase-9-dependent pathway in mouse and human osteosarcoma cells by a new nitroxyl spin-labeled derivative of podophyllotoxin

Previous study has found that a new nitroxyl spin-labeled derivative of podophyllotoxin, 4-[4″-(2″,2″,6″,6″-tetramethyl-1″-piperidinyloxy)amino]-4′-demethyl-epipodophyllotoxin (GP7), can induce apoptosis in human leukemia cells. However, there have been no studies about the effects of GP7 on osteosarcoma (OS) cells. Here, we observed the anti-OS effects of GP7 in mouse and human OS cells with the comparison of etoposide. GP7 and etoposide inhibited the proliferation of a panel of mouse and human OS cells in a concentration- or time-dependent manner, and the inhibitory effect of GP7 on the proliferation of mouse LM8 or human U2OS cells was 1.28- or 1.35-fold higher than that of etoposide. GP7 or etoposide augmented the anti-OS effects of methotrexate, adriamycin, cisplatin, or their combination, and the combined inhibitory effects of GP7 with MTX on the proliferation of LM8 cells was higher than those of etoposide with MTX. GP7 arrested the cell cycle in S phase but etoposide in G₂/M phase. GP7 or etoposide induced sub-G₁ peak, apoptotic DNA fragmentation, activations of caspase-3, -8, -9, and DNA fragmentation factor, downregulation of Bcl-2 and Bcl-xL, upregulation of Bax and Bak, and cytochrome-c release from mitochondria in both mouse and human OS cells. GP7 or etoposide also induced endonuclease G translocation from mitochondria into cytosol in mouse cells. GP7- or etoposide-induced apoptotic DNA fragmentation of human OS cells was inhibited by the pan caspase inhibitor and caspase-9 inhibitor, not by caspase-8 inhibitor whereas it was not inhibited by the pan caspase inhibitor in mouse OS cells. Our findings indicate that GP7 is effective against mouse and human OS cells in vitro. The apoptotic DNA fragmentation in mouse OS cells may be mediated by caspase-independent pathway with the involvement of endonuclease G whereas in human OS cells by caspase-9-dependent pathway downstream of the cytochrome-c-initiated caspase cascade.     

Sulfuretin from heartwood of Rhus verniciflua triggers apoptosis through activation of Fas, Caspase-8, and the mitochondrial death pathway in HL-60 human leukemia cells

Sulfuretin, a flavonoid isolated from heartwood of Rhus verniciflua, has been reported to have anti-cancer activities but the underlying molecular mechanism was not clear. In this study, sulfuretin induced apoptosis by activating caspases-8, -9, and -3 as well as cleavage of poly(ADP-ribose) polymerase. Furthermore, treatment with sulfuretin caused mitochondrial dysfunctions, including the loss of mitochondrial membrane potential (ΔΨ(m)), the release of cytochrome c to the cytosol, and the translocations of Bax and tBid. Sulfuretin also activated the extrinsic apoptosis pathway, that is, it increased the expressions of Fas and FasL, the activation of caspase-8, and the cleavage of Bid. Furthermore, blocking the FasL-Fas interaction with NOK-1 monoclonal antibody prevented the sulfuretin-induced apoptosis. The therapeutical effect of sulfuretin in leukemia is due to its potent apoptotic activity through the extrinsic pathway driven by a Fas-mediated caspase-8-dependent pathway.     

Trichosanthin induced apoptosis in HL-60 cells via mitochondrial and endoplasmic reticulum stress signaling pathways

Trichosanthin (TCS), a traditional Chinese medicine, exerts antitumor activities by inducing apoptosis in many different tumor cell lines. However, the mechanisms remain obscure. The present study focused on various caspase pathways that may be involved in TCS-induced apoptosis in leukemia HL-60 cells. Key caspases in both intrinsic and extrinsic pathways including caspase-8, -9 and -3 were activated upon TCS treatment. Additionally, TCS treatment induced upregulation of BiP and CHOP and also activated caspase-4, which for the first time strongly supported the involvement of endoplasmic reticulum stress pathway in TCS-induced apoptosis. Interestingly, although caspase-8 was activated, Fas/Fas ligand pathway was not involved as evidenced by a lack of induction of Fas or Fas ligand and a lack of inhibitory effect of anti-Fas blocking antibody on TCS-induced apoptosis. Instead, caspase-8 was activated in a caspase-9 and -4 dependent manner. The involvement of mitochondria was demonstrated by the reduction of mitochondrial membrane potential and release of cytochrome c and Smac besides the activation of caspase-9. Further investigation confirmed that caspase-3 was the major executioner caspase downstream to caspase-9, -4 and -8. Taken together, our results suggested that TCS-induced apoptosis in HL-60 cells was mainly mediated by mitochondrial and ER stress signaling pathways via caspase-3.     

Effects of Chronic and Acute Zinc Supplementation on Myocardial Ischemia-Reperfusion Injury in Rats

A ginseng polysaccharide was extracted, purified, and modified by nitric acid-selenious acid (HNO₃-H₂SeO₃) method to yield one selenylation-modified polysaccharide (sGP). We reported for the first time the anticancer potential of sGP on the human promyelocytic leukemia HL-60 cell line and evaluated its relevant underlying mechanism. Our results showed that sGP markedly inhibited the growth of HL-60 cells via induction of apoptosis. The event of apoptosis was accompanied by the formation of apoptotic bodies; the release of cytochrome c; loss of mitochondrial membrane potential; and activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. In addition, western blot analysis showed that sGP inhibited antiapoptotic Bcl-2 protein expression and increased proapoptotic Bax protein expression in cells under identical conditions. Together, our study suggests that sGP induces apoptosis of HL-60 cells through the mitochondrial-dependent pathway.     

Induction of apoptosis by collinin from Zanthoxylum schinifolium is mediated via mitochondrial pathway in human Jurkat T cells

Collinin, which was isolated from the leaves of Zanthoxylum schinifolium, could exert cytotoxic effect on various human tumor cells with IC₅₀ values in the range of 38.1–111.6 μM, whereas the IC₅₀ value for human normal mammary epithelial MCF-10A cells was 124.4 μM. To examine the contribution of apoptosis to the cytotoxicity of collinin toward tumor cells, collinin-induced apoptotic events of Jurkat T cells transfected with vector (JT/Neo) were compared with those of Jurkat T cells transfected with Bcl-2 gene (JT/Bcl-2). Treatment of JT/Neo cells with collinin (30–60 μM) resulted in induction of sub-G₁ peak representing apoptotic cells along with activation of Bak and Bax, mitochondrial membrane potential (Δψ_m) loss, activation of caspase-9, -3, -8, and -7, degradation of PARP, and DNA fragmentation dose-dependently, but these apoptotic events were abrogated by overexpression of Bcl-2, which could prevent the induced activation of Bak and Bax, and subsequent mitochondrial damage. Under these conditions, necrosis was not accompanied. Pretreatment of JT/Neo cells with the pan-caspase inhibitor z-VAD-fmk completely blocked collinin-induced apoptotic sub-G₁ cells and caspase cascade activation, whereas it failed to suppress Bak activation and Δψ_m loss. Neither FADD-deficiency nor caspase-8-deficiency affected the susceptibility of Jurkat T cells to collinin-induced cytotoxicity and apoptotic cell death. These results demonstrate that the apoptogenic activity of collinin was mediated by the intrinsic mitochondrial apoptotic pathway which was preceded by activation of pro-apoptotic multidomain Bcl-2 family members Bak and Bax, mitochondrial damage, and resultant activation of caspase cascade, leading to PARP degradation, which could be regulated by Bcl-2.     

Bcl-3 regulates UVB-induced apoptosis

B cell leukemia-3 (Bcl-3) has been defined as an anti-apoptotic gene; however, the exact mechanisms through which Bcl-3 influences apoptosis have been elusive. To determine the specific role of Bcl-3 in apoptosis, we evaluated the effect of its silencing on the expression of proteins involved in either the extrinsic or intrinsic apoptotic pathways induced by ultraviolet light B-mediated DNA damage. We found that, in Bcl-3-silenced cells, caspase-3, caspase-8 and caspase-9 activation is accelerated and tBid mitochondrial content is increased. It is important to note that, although mitochondrial Smac levels were reduced after UV exposure, the rate of reduction was slightly higher in Bcl-3 silenced cells than in control cells. Additionally, p53 levels diminished in Bcl-3 silenced cells compared to control cells, as did those of DNA-PK, a DNA repair protein. Altogether, our data indicate that Bcl-3 protects cells from apoptosis by regulating both apoptotic pathways.     

Cordyceps sinensis mycelium extract induces human premyelocytic leukemia cell apoptosis through mitochondrion pathway

This study was to identify the signaling pathways for the induction of HL-60 cell apoptosis by Cordyceps sinensis mycelium extract (CSME). CSME at 25 mug/ml induced nuclear fragmentation and DNA degradation, two hallmark events of apoptosis, in the HL-60 cells within 12-24 hrs of treatment. Concomitantly, several major events in the mitochondrial signal pathway occurred, including the loss of MTP (DeltaPsi(m)), cytochrome c release into the cytoplasm, the decrease in Bcl-2 protein level, the translocation of Bax protein from cytoplasm into mitochondria, and the activation of caspase-2, -3, and -9, but caspase-8, the initiator caspase in the death receptor pathway, was not activated. These results suggest that CSME induces apoptosis in HL-60 cell through the mitochondrial pathway rather than the death receptor pathway.     

In vitro cytotoxic potential of Polyalthia longifolia on human cancer cell lines and induction of apoptosis through mitochondrial-dependent pathway in HL-60 cells

Polyalthia longifolia is a lofty evergreen tree found in India and Sri Lanka. We are reporting first time the anticancer potential of P. longifolia leaves extract (A001) and its chloroform fraction (F002). Both inhibited cell proliferation of various human cancer cell lines in which colon cancer cells SW-620 showed maximum inhibition with IC(50) value 6.1 microg/ml. Furthermore, F002 induce apoptosis in human leukemia HL-60 cells as measured by several biological end points. F002 induce apoptotic bodies formation, DNA ladder, enhanced annexin-V-FITC binding of the cells, increased sub-G(0) DNA fraction, loss of mitochondrial membrane potential (DeltaPsi(mt)), release of cytochrome c, activation of caspase-9, caspase-3, and cleavage of poly ADP ribose polymerase (PARP) in HL-60 cells. All the above parameters revealed that F002-induced apoptosis through the mitochondrial-dependent pathway in HL-60 cells.     

Induction of apoptosis by carbazole alkaloids isolated from Murraya koenigii.

In the current study, we isolated 10 carbazole alkaloids from the plant species Murraya koenigii (Rutaceae), and examined their effects on the growth of the human leukemia cell line HL-60. Three carbazole alkaloids, mahanine (6), pyrayafoline-D (7) and murrafoline-I (9), showed significant cytotoxicity against HL-60 cells. Fluorescence microscopy with Hoechst 33342 staining revealed that the percentage of apoptotic cells with fragmented nuclei and condensed chromatin was increased in a time-dependent manner after treatment with each alkaloid. Interestingly, each carbazole alkaloid induced the loss of mitochondrial membrane potential. In addition, both caspase-9 and caspase-3 were also time-dependently activated upon treatment with the alkaloids. Caspase-9 and caspase-3 inhibitors suppressed apoptosis induced by these alkaloids. The results suggest that these three alkaloids induced apoptosis in HL-60 cells through activation of the caspase-9/caspase-3 pathway, through mitochondrial dysfunction.     

Midazolam induces cellular apoptosis in human cancer cells and inhibits tumor growth in xenograft mice

Midazolam is a widely used anesthetic of the benzodiazepine class that has shown cytotoxicity and apoptosisinducing activity in neuronal cells and lymphocytes. This study aims to evaluate the effect of midazolam on growth of K562 human leukemia cells and HT29 colon cancer cells. The in vivo effect of midazolam was investigated in BALB/c-nu mice bearing K562 and HT29 cells human tumor xenografts. The results show that midazolam decreased the viability of K562 and HT29 cells by inducing apoptosis and S phase cell-cycle arrest in a concentration-dependent manner. Midazolam activated caspase-9, capspase-3 and PARP indicating induction of the mitochondrial intrinsic pathway of apoptosis. Midazolam lowered mitochondrial membrane potential and increased apoptotic DNA fragmentation. Midazolam showed reactive oxygen species (ROS) scavenging activity through inhibition of NADPH oxidase 2 (Nox2) enzyme activity in K562 cells. Midazolam caused inhibition of pERK1/2 signaling which led to inhibition of the anti-apoptotic proteins Bcl-X_L and XIAP and phosphorylation activation of the pro-apoptotic protein Bid. Midazolam inhibited growth of HT29 tumors in xenograft mice. Collectively our results demonstrate that midazolam caused growth inhibition of cancer cells via activation of the mitochondrial intrinsic pathway of apoptosis and inhibited HT29 tumor growth in xenograft mice. The mechanism underlying these effects of midazolam might be suppression of ROS production leading to modulation of apoptosis and growth regulatory proteins. These findings present possible clinical implications of midazolam as an anesthetic to relieve pain during in vivo anticancer drug delivery and to enhance anticancer efficacy through its ROS-scavenging and pro-apoptotic properties.