Xuelian Zheng

Luteolin enhances TNF-related apoptosis-inducing ligand's anticancer activity in a lung cancer xenograft mouse model.

Sensitization of cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by luteolin has been suggested by in vitro studies. However, no in vivo experiment has been reported to validate the potentiation effect of luteolin on TRAILs anticancer activity. In this report, we first confirmed that luteolin potentiates TRAIL-induced cytotoxicity in A549 cells and HeLa cells in association with increased activation of apoptosis. Then we performed an in vivo experiment with a non-small cell lung cancer xenograft mouse model, which showed for the first time that the in vivo anticancer activity of TRAIL was greatly enhanced by luteolin. Compared with that in untreated control or treatment with TRAIL or luteolin alone, inhibition of tumor growth and apoptotic cell death in xenograft tumors were significantly increased in animals receiving combination treatment with TRAIL and luteolin. Data from this study thus provide strong in vivo evidence supporting that luteolin is a potential sensitizer for TRAIL in anticancer therapy.

Wogonin enhances antitumor activity of tumor necrosis factor-related apoptosis-inducing ligand in vivo through ROS-mediated downregulation of cFLIPL and IAP proteins.

Combination of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) with other agents is a promising strategy to overcome TRAIL resistance in malignant cells. Wogonin, a flavonoid originated from Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL’s antitumor activity in vivo has never been studied. In this study, the effect of combination of TRAIL and wogonin was tested in a non-small-cell lung cancer xenografted tumor model in nude mice. Consistent with the in vitro study showing that wogonin sensitized A549 cells to TRAIL-induced apoptosis, wogonin greatly enhanced TRAIL-induced suppression of tumor growth, accompanied with increased apoptosis in tumor tissues as determined by TUNEL assay. The expression levels of antiapoptotic proteins including long form of cellular FLICE-like inhibitory protein (cFLIPL), X-linked inhibitor of apoptosis protein (XIAP), and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues after co-treatment with wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated by proteasomal degradation that involved intracellular reactive oxygen species (ROS), because wogonin robustly induced ROS accumulation and ROS scavengers butylated hydroxyanisole (BHA) and N-acetyl-l-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins in cells co-treated with wogonin and TRAIL. These results show for the first time that wogonin enhances TRAIL’s antitumor activity in vivo, suggesting this strategy has an application potential for clinical anticancer therapy.

Wogonin potentiates cisplatin-induced cancer cell apoptosis through accumulation of intracellular reactive oxygen species

Chemoresistance to cisplatin is a major limitation of cisplatin-based chemotherapy in the clinic. The combination of cisplatin with other agents has been recognized as a promising strategy to overcome cisplatin resistance. Previous studies have shown that wogonin (5,7-dihydroxy-8-methoxyflavone), a flavonoid isolated from the root of the medicinal herb Scutellaria baicalensis Georgi, sensitizes cancer cells to chemotheraputics such as etoposide, adriamycin, tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and TNF. However, the effect of wogonin on cisplatin-induced cytotoxicity has not been previously reported. In this study, the non-small cell lung cancer cell line A549 and the cervical cancer cell line HeLa were treated with wogonin or cisplatin individually or in combination. It was found for the first time that wogonin is able to sensitize cisplatin-induced apoptosis in both A549 cells and HeLa cells as indicated by the potentiation of activation of caspase-3, and cleavage of the caspase-3 substrate PARP in wogonin and cisplatin co-treated cells. Importantly, wogonin robustly induced H2O2 accumulation in these cells, which substantially contributes to the sensitization of cisplatin cytotoxicity by wogonin, as two reactive oxygen species scavengers, butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC), significantly suppressed the potentiated cytotoxicity caused by wogonin and cisplatin co-treatment. The results from this study provide important new evidence supporting the potential use of wogonin as a cisplatin sensitizer for cancer therapy.

Concurrent blockade of the NF-κB and Akt pathways potently sensitizes cancer cells to chemotherapeutic-induced cytotoxicity

Nuclear factor-kappaB (NF-kappaB) and Akt are two major cell survival pathways that are often constitutively activated and can be further stimulated by chemotherpeutics in cancer cells. Although individually targeting the NF-kappaB or Akt has been reported to sensitize caner therapy, the effectiveness of concurrent blocking these two pathways for chemosensitizing of cancer cells to genotoxic therapeutics has not been investigated. In the present study, we investigate the activation of the NF-kappaB and Akt pathways by two frontline anticancer drugs cisplatin and etopside in a variety of cancer cell lines. The effects of blocking these two survival pathways individually or concurrently on cisplatin- or etopside-induced cytotoxicity were detected. The results show that cisplatin and etopside activate both NF-kappaB and Akt in cancer cells. Blockade of either of these pathways with chemical inhibitors or siRNA moderately sensitized cancer cells to cisplatin- or etopside-induced cytotoxicity. Strikingly, much more effective potentiation of cytotoxicity to these anticancer drugs was achieved when NF-kappaB and Akt were concurrently blocked. These data suggest that NF-kappaB and Akt cooperatively attenuate therapeutic-induced cytotoxicity and concurrently blocking these pathways is an effective strategy for improving the anticancer efficacy of therapeutics.

Critical role of CD40-mediated autocrine tumor necrosis factor-alpha in potentiation of cisplatin-induced cytotoxicity in cancer cells.

Activation of CD40, a member of the tumor necrosis factor receptor (TNF‐R) family, results in growth inhibition or apoptosis in some tumor cells, making CD40 a potential antitumor therapeutic target. Although it is known that CD40 is able to induce tumor necrosis factor‐alpha (TNF‐α) secretion and potentiate cisplatin’s anticancer activity, whether TNF‐α induction is involved in sensitizing cisplatin by CD40 has not been addressed. In this report, we provide evidence substantiating an important role of autocrine TNF‐α in potentiation of cisplatin‐induced apoptosis by recombinant soluble CD40 ligand (rsCD40L) in different human cancer cell lines. Activation of CD40 by rsCD40L induces two phases of autocrine TNF‐α: the rapid early phase involving p38 MAP kinase and the robust and persistent late phase through enhanced tnf‐α gene transcription. Blocking TNF‐α with either a specific TNFR1 siRNA or a neutralizing anti‐TNF‐α antibody dramatically attenuated the potentiation effect of rsCD40L on cisplatin‐induced cancer cell death. These results reveal an important role of TNF‐α induction in CD40’s chemosensitization activity and suggest that modulating TNF‐α autocrine from cancer cells is an effective option for increasing the anticancer value of chemotherapeutics such as cisplatin. (Cancer Sci 2012; 103: 197–202)

Concurrent blockade of NF-κB and Akt pathways potentiates cisplatin's antitumor activity in vivo.

Nuclear factor-&kgr;B (NF-&kgr;B) and Akt are two major cell-survival pathways that are often constitutively activated in cancer cells. It has been established that these two pathways contribute substantially toward the chemoresistance of cancer cells. Our previous study has demonstrated that NF-&kgr;B and Akt cooperatively blunt cytotoxicity induced by cisplatin or etopside in different types of cancer cells in vitro, indicating that the concurrent blocking of these pathways may effectively improve the anticancer efficacy of anticancer therapeutics. In this study, we further investigated the effect of concurrent blockade of NF-&kgr;B and Akt on the anticancer activity of cisplatin in vivo in a xenograft tumor model. The NF-&kgr;B and Akt pathways in the A549 lung cancer cells were blocked individually or concurrently by the stable transfection of plasmids expressing short hairpin RNAs that target Akt1 and I&kgr;B kinase &bgr;. The resultant cells with concurrent NF-&kgr;B and Akt blockade were significantly more sensitive to cisplatin-induced cell death in vitro. Consistently, tumors derived from cells with the concurrent blockade of NF-&kgr;B and Akt were much more sensitive to cisplatin compared with those derived from cells with individual blockage of NF-&kgr;B or Akt in a nude mouse xenograft tumor model. Apoptosis was significantly enhanced in the double pathway-suppressed and cisplatin-treated tumors. These results show for the first time that the concurrent blockage of the NF-&kgr;B and Akt pathways cooperatively potentiates the antitumor activity of cisplatin in vivo, indicating that this strategy may be potentially useful for clinical anticancer therapy.

Reversal of multidrug resistance in vitro and in vivo by 5- N -formylardeemin, a new ardeemin derivative

Because multidrug resistance (MDR) is a serious impediment to the use of chemotherapy in treating cancer patients, great efforts have been made to search for effective MDR-reversing agents. We have developed a brand new synthetic ardeemin derivative, 5-N-formylardeemin, and investigated the activity of which in reversing MDR in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin, vincristine through potentiation of apoptosis in both MCF-7-R and A549-R at relatively noncytotoxic concentrations in vitro. Mechanistic studies showed that 5-N-formylardeemin inhibited the expression of MDR-1 (P-gp) and increased the intracellular accumulation of cytotoxic drugs in the MDR cells, suggesting that 5-N-formylardeemin reverses MDR activities through inhibiting MDR-1 expression. Interestingly, 5-N-formylardeemin also sensitized the parent wild-type cancer cells toward these chemotherapeutic agents to various extents. Importantly, in vivo studies demonstrated that 5-N-formylardeemin significantly improved the therapeutic effects of doxorubicin in nude mice bearing A549-R xenografts, which was associated with reduced expression of MDR-1 protein level and increased apoptosis in tumor tissues. These results underscore 5-N-formylardeemin as a potential sensitizer for chemotherapy against multidrug resistant cancers.

Histone H4 expression is cooperatively maintained by IKKβ and Akt1 which attenuates cisplatin-induced apoptosis through the DNA-PK/RIP1/IAPs signaling cascade.

While chromatin remodeling mediated by post-translational modification of histone is extensively studied in carcinogenesis and cancer cell’s response to chemotherapy and radiotherapy, little is known about the role of histone expression in chemoresistance. Here we report a novel chemoresistance mechanism involving histone H4 expression. Extended from our previous studies showing that concurrent blockage of the NF-κB and Akt signaling pathways sensitizes lung cancer cells to cisplatin-induced apoptosis, we for the first time found that knockdown of Akt1 and the NF-κB-activating kinase IKKβ cooperatively downregulated histone H4 expression, which increased cisplatin-induced apoptosis in lung cancer cells. The enhanced cisplatin cytotoxicity in histone H4 knockdown cells was associated with proteasomal degradation of RIP1, accumulation of cellular ROS and degradation of IAPs (cIAP1 and XIAP). The cisplatin-induced DNA-PK activation was suppressed in histone H4 knockdown cells, and inhibiting DNA-PK reduced expression of RIP1 and IAPs in cisplatin-treated cells. These results establish a novel mechanism by which NF-κB and Akt contribute to chemoresistance involving a signaling pathway consisting of histone H4, DNA-PK, RIP1 and IAPs that attenuates ROS-mediated apoptosis, and targeting this pathway may improve the anticancer efficacy of platinum-based chemotherapy.

Abstract 3724: Wogonin enhances tumor necrosis factor-related apoptosis-inducing ligand's antitumor activity in vivo through H2O2-mediated downregulation of X-linked inhibitor of apoptosis protein and cellular inhibitor of apoptosis protein 1 and 2

Numerous malignant cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity, limiting value of TRAIL as an antitumor therapeutics. Combination of TRAIL with other agents is a promising strategy to overcome TRAIL resistance. Wogonin (5, 7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of the medicinal herb Scutellaria baicalensis Georgi, has been shown to enhance TRAIL-induced apoptosis in malignant cells in in vitro studies. However, whether wogonin enhances TRAIL9s antitumor activity in vivo has never been studied. In this study, we first found that wogonin sensitized a variety of cancer cells including A549, HeLa and SKOV3 to TRAIL-induced apoptosis, as indicated by increased annexin V staining and enhanced activation of caspase-8 and -3 in TRAIL and wogonin cotreated cells, whereas having marginal effect on non-transformed bronchial epithelial cells and cervical epithelial cells. Then the effect of combination of TRAIL and wogonin was tested in a xenografted tumor model with A549 cells in nude mice. The results show that wogonin greatly enhanced TRAIL-induced suppression of tumor growth, which was accompanied with increased apoptosis in tumor tissues determined by a terminal deoxynucleotidyl-transferase -mediated dUTP nick-end labeling (TUNEL) assay. The protein levels of X-linked inhibitor of apoptosis protein (XIAP) and cellular inhibitor of apoptosis protein 1 and 2 (cIAP-1 and cIAP-2) were markedly reduced in both cultured cells and xenografted tumor tissues with co-treatment of wogonin and TRAIL. The down-regulation of these antiapoptotic proteins was likely mediated through proteasomal degradation and involved intracellular H 2 O 2 , as wogonin robustly induced H 2 O 2 accumulation in A549 cells and the reactive oxygen species scavengers butylated hydroxyanisole (BHA) and N-acetyl-L-cysteine (NAC) and the proteasome inhibitor MG132 restored the expression of these antiapoptotic proteins and suppressed cytotoxicity in cells co-treated with wogonin and TRAIL. These results provide important in vivo evidence supporting the application of wogonin as TRAIL sensitizer for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3724. doi:1538-7445.AM2012-3724