Efficacy Of Chemotherapeutic Drugs Research Articles

Interferon-β lipofection I. Increased efficacy of chemotherapeutic drugs on human tumor cells derived monolayers and spheroids

We evaluated the effect of hIFNβ gene transfer alone or in combination with different antineoplastic drugs commonly used in cancer treatment. Five human tumor-derived cell lines were cultured as monolayers and spheroids. Four cell lines (Ewing sarcomas EW7 and COH, melanoma M8 and mammary carcinoma MCF-7) were sensitive to hIFNβ gene lipofection. Although this effect appeared in both culture configurations, spheroids showed a relative multicellular resistance (insensitive colon carcinoma HT-29 excluded). EW7 and M8 hIFNβ-expressing cells were exposed to different concentrations of bleomycin, bortezomib, carboplatin, doxorubicin, etoposide, methotrexate, paclitaxel and vincristine in both configuration models. In chemotherapy-sensitive EW7 monolayers, the combination of hIFNβ gene and antineoplastic drugs displayed only additive or counteractive (methotrexate) effects, suggesting that cytotoxic mechanisms triggered by hIFNβ gene lipofection could be saturating the signaling pathways. Conversely, in chemotherapy-resistant EW7 spheroids or M8 cells, the combination of hIFNβ with drugs that mainly operate at the genotoxic level (doxorubicin, methotrexate and paclitaxel) presented only additive effects. However, drugs that also increase pro-oxidant species can complement the antitumor efficacy of the hIFNβ gene and clearly caused potentiated effects (bleomycin, bortezomib, carboplatin, etoposide and vincristine). The great bystander effect induced by hIFNβ gene lipofection could be among the main causes of its effectiveness, because only 1 or 2% of EW7 or M8 hIFNβ-expressing cells killed more than 60 or 80% of cell population, respectively.

Drug resistance associates with activation of Nrf2 in MCF‐7/DOX cells, and wogonin reverses it by down‐regulating Nrf2‐mediated cellular defense response

Acquired resistance to doxorubicin (DOX) is a serious therapeutic problem in breast cancer patients. In this study, we investigated whether nuclear factor erythroid 2-related factor 2 (Nrf2) was associated with drug resistant in DOX resistant MCF-7 (MCF-7/DOX) cells, and if wogonin, a flavonoid isolated from the root of Scutellaria baicalensis Georgi, could reverse drug resistance in MCF-7/DOX cells. We found that the endogenous expression of Nrf2 as well as its target proteins heme oxygenase-1 (HO-1), NADP(H):quinone oxidoreductase (NQO) in MCF-7/DOX cells was higher than that in MCF-7 cells. Tert-butylhydroquinone treatment increased the expression Nrf2, HO-1, and NQO-1, and enhanced resistance of MCF-7 cells to DOX. Similarly, intracellular Nrf2 protein level was significantly decreased in MCF-7/DOX cells and DOX resistance was partially reversed by Nrf2 siRNA. Wogonin downregulated the Nrf2-dependent response and partly reversed DOX resistance in MCF-7/DOX cells. These results suggested that activation of Nrf2 was associated with drug resistance in MCF-7/DOX cells. Wogonin reversed drug resistance and its reversal mechanism might be due to the suppression of Nrf2 signaling pathway, indicating the feasibility of using Nrf2 inhibitors to increase efficacy of chemotherapeutic drugs.

Axitinib Targeted Cancer Stemlike Cells to Enhance Efficacy of Chemotherapeutic Drugs via Inhibiting the Drug Transport Function of ABCG2

Stemlike cells have been isolated by their ability to efflux Hoechst 33342 dye and are called the side population (SP). We evaluated the effect of axitinib on targeting cancer stemlike cells and enhancing the efficacy of chemotherapeutical agents. We found that axitinib enhanced the cytotoxicity of topotecan and mitoxantrone in SP cells sorted from human lung cancer A549 cells and increased cell apoptosis induced by chemotherapeutical agents. Moreover, axitinib particularly inhibited the function of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) and reversed ABCG2-mediated multidrug resistance (MDR) in vitro. However, no significant reversal effect was observed in ABCB1-, ABCC1- or lung resistance-related protein (LRP)-mediated MDR. Furthermore, in both sensitive and MDR cancer cells axitinib neither altered the expression of ABCG2 at the mRNA or protein levels nor blocked the phosphorylation of AKT and extracellular signal-regulated kinase (ERK)1/2. In nude mice bearing ABCG2-overexpressing S1-M1-80 xenografts, axitinib significantly enhanced the antitumor activity of topotecan without causing additional toxicity. Taken together, these data suggest that axitinib particularly targets cancer stemlike cells and reverses ABCG2-mediated drug resistance by inhibiting the transporter activity of ABCG2.

Su1869 DCAMKL-1 Regulates Pluripotency Factors Oct4, SOX2 and KLF4 via miR-145 MicroRNA Dependent Mechanism

Our current study investigated the effect of PHLPP expression on the efficacy of chemotherapeutic drugs in colon cancer cells. Both PHLPP isoforms, PHLPP1 and PHLPP2, were either knocked down or overexpressed in SW480 and HT29 human colon cancer cells, and the rate of cell proliferation and apoptosis were measured upon drug treatment. Specifically, the cells were treated with different concentrations of oxaliplatin, PI3K inhibitor (LY294002), or rapamycin for 48 hours and cell proliferation was determined using MTS assays. Our results showed that knockdown of both PHLPP isoforms resulted in a decrease in the sensitivity to all drugs tested, whereas overexpression of PHLPP enhanced the anti-proliferation effect of these drugs. Moreover, we assessed the effect of the chemotherapy drugs in inducing cell death by monitoring the appearance of apoptotic markers including cleaved PARP and Caspase-3. Similarly, knockdown of both PHLPP isoforms rendered the cells resistant to apoptosis upon drug treatment. In summary, we have identified PHLPP as a critical factor in determining the drug sensitivity in the chemotherapeutic treatment of colon cancer. Adding yet another novel function of PHLPP and reasoning for the possible use of PHLPP as a therapeutic target in colon cancer.

Su1871 Lactate Dehydrogenase-a Induction as an Early Metabolomic Marker of Colon Carcinogenesis: Potential Target for Chemoprevention

Our current study investigated the effect of PHLPP expression on the efficacy of chemotherapeutic drugs in colon cancer cells. Both PHLPP isoforms, PHLPP1 and PHLPP2, were either knocked down or overexpressed in SW480 and HT29 human colon cancer cells, and the rate of cell proliferation and apoptosis were measured upon drug treatment. Specifically, the cells were treated with different concentrations of oxaliplatin, PI3K inhibitor (LY294002), or rapamycin for 48 hours and cell proliferation was determined using MTS assays. Our results showed that knockdown of both PHLPP isoforms resulted in a decrease in the sensitivity to all drugs tested, whereas overexpression of PHLPP enhanced the anti-proliferation effect of these drugs. Moreover, we assessed the effect of the chemotherapy drugs in inducing cell death by monitoring the appearance of apoptotic markers including cleaved PARP and Caspase-3. Similarly, knockdown of both PHLPP isoforms rendered the cells resistant to apoptosis upon drug treatment. In summary, we have identified PHLPP as a critical factor in determining the drug sensitivity in the chemotherapeutic treatment of colon cancer. Adding yet another novel function of PHLPP and reasoning for the possible use of PHLPP as a therapeutic target in colon cancer.

Abstract 825: Wnt/beta-catenin signaling regulates the expression of O6-methylguanine DNA-methyltransferase in cancer cells: a new strategy to overcome resistance for DNA alkylators in cancer therapy

Abstract Background: A number of DNA-damaging agents attack the O6 position on guanine and thereby form the most potent cytotoxic DNA adducts known. The DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) encoded by the gene O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression correlates inversely with sensitivity to agents that form O6-alkylguanine adducts, such as temozolamide. It is therefore of great interest to find agents that induce MGMT deficiency, increase the sensitivity and possible overcoming resistance to alkylating chemotherapeutic agents. Methods: Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/beta-catenin activity and MGMT expression. We used Western blot, Real-Time quantitative PCR (Q-PCR), siRNA knockdown and cDNA overexpression of beta-catenin, MGMT promotor reporter plasmids and cells with inducible siRNAs targeting beta-catenin to study beta-catenin mediated regulation of MGMT. The correlation between Wnt activity and MGMT expression was also investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. Compounds targeting Wnt signaling was investigated in combination with temolzolamide in vitro and in vivo. Results: MGMT expression level was shown be correlated to Wnt signaling activation both in primary tumors and cell lines of different origins. Proinflammatory prostaglandin E2 activates the Wnt/beta-catenin signaling and increase MGMT expression. Transfection experiments and cells with inducible siRNAs revealed that beta-catenin directly regulates MGMT expression via Tcf/LEF binding. Wnt inhibiting drugs and compounds potentiates the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression in vitro and in vivo. Conclusions: Our data demonstrate that MGMT is a direct Wnt/beta-catenin target, and agents that inhibit Wnt signalling reduces the transcription of MGMT through a prostaglandin E2-Wnt/beta-catenin route and thus increases the sensitivity to temozolomide. This provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. The data also suggest that Wnt/beta-catenin is an important target for therapeutic interventions. 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 825. doi:1538-7445.AM2012-825

Chemokine and Chemokine Receptor Expression in Kidney Tumors: Molecular Profiling of Histological Subtypes and Association With Metastasis

Molecular characterization of renal cell carcinoma may help differentiate benign oncocytoma from malignant renal cell carcinoma subtypes and predict metastasis. Chemokines, eg IL-8 and chemokine receptors such as CXCR4 and 7, promote inflammation and metastasis. SDF-1 is a CXCR4 and 7 ligand with 6 known isoforms. We evaluated the expression of these chemokines and chemokine receptors in kidney specimens. Using quantitative polymerase chain reaction we measured mRNA levels of IL-8, CXCR4 and 7, and SDF1 isoforms α, β and γ in a total of 166 specimens from 86 patients, including 86 tumor samples and 80 matched normal kidney samples. Mean ± SD followup was 18.9 ± 12 months (median 19.5). Renal cell carcinoma specimens included the clear cell, papillary and chromophobe subtype in 65, 10 and 5 cases, respectively, and oncocytoma in 6. A total of 17 cases were positive for metastasis. Median CXCR4 and 7, and SFD1-γ levels were increased twofold to tenfold. SDF1-α and β were unchanged or lower in clear cell renal cell carcinoma and papillary tumors than in normal tissue. Median SDF1-γ, IL-8, and CXCR4 and 7 were increased threefold to fortyfold in chromophobe tumors compared to oncocytoma. CXCR4 and 7 were increased in tumors less than 4 cm (mean 3,057 ± 2,230 and 806 ± 691) compared to oncocytoma (336 ± 325 and 201 ± 281, respectively, p ≤0.016). On multivariate analysis CXCR4 (p = 0.01), CXCR7 (p = 0.02) and SDF1-β (p = 0.005) were independently associated with metastasis. Combined CXCR7 plus SDF1-α and CXCR7 plus IL-8 markers showed the highest sensitivity (71% to 81%) and specificity (75% to 80%) of all individual or combined markers. Chemokines and chemokine receptors differentiate renal cell carcinoma and oncocytoma. Combined SDF1-α plus CXCR7 and IL-8 plus CXCR7 markers have about 80% accuracy for predicting renal cell carcinoma metastasis.

Apatinib (YN968D1) enhances the efficacy of conventional chemotherapeutical drugs in side population cells and ABCB1-overexpressing leukemia cells

P-glycoprotein (P-gp, ABCB1) overexpression and enrichment of stem-like cells are linked to poor prognosis in tumor patients. In this study, we investigated the effect of apatinib, an oral multi-targeted tyrosine kinase inhibitor (TKI) on enhancing the efficacy of conventional anticancer drugs in side population (SP) cells and ABCB1-overexpressing leukemia cells in vitro, in vivo and ex vivo. Our results showed that apatinib significantly enhanced the cytotoxicity and cell apoptosis induced by doxorubicin in SP cells sorted from K562 cells. Furthermore, apatinib also strongly reversed multidrug resistance (MDR) in K562/ADR cells, and the primary leukemia blasts overexpressing ABCB1 while showed no synergistic interactions with chemotherapeutic agents in MRP1-, MRP4-, MRP7- and LRP-overexpressing cells. Apatinib treatment markedly increased the intracellular accumulation of doxorubicin and rhodamine 123 in K562/ADR cells and the accumulation of rhodamine 123 in the primary leukemia blasts with ABCB1 overexpression. Apatinib stimulated the ATPase activity of P-gp in a dose-dependent manner but did not alter the expression of ABCB1 at both mRNA and protein levels. The phosphorylation level of AKT and ERK1/2 remained unchanged after apatinib treatment in both sensitive and MDR cells. Importantly, apatinib significantly enhanced the antitumor activity of doxorubicin in nude mice bearing K562/ADR xenografts. Taken together, our results suggest that apatinib could target to SP cells and ABCB1-overexpressing leukemia cells to enhance the efficacy of chemotherapeutic drugs. These findings should be useful for the combination of apatinib and chemotherapeutic agents in the clinic.

Immunostimulatory effects on B cells by the chemotherapeutic drug Doxorubicin

The efficacy of chemotherapeutic drugs has traditionally been evaluated by their ability to exhibit a direct cytotoxic effect on malignant cells. Recently, it has been reported that certain of these drugs can also enhance the antitumor efficacy and immunotherapy response through their capacity to modulate innate and adaptive immunity [1]. Our research group has earlier utilized a treatment protocol based on the principle that tumor reactive T cells can be isolated from sentinel lymph nodes, expanded in vitro and then be given back to the patient as an autologous transfusion [2]. B cells are the most abundant APC in lymph nodes, and it is therefore of interest to study the effects of chemotherapeutic drugs to B cell antigen presenting function.

Abstract LB-209: Expression of O6-methylguanine DNA-methyltransferase is regulated by Wnt/β-catenin signalling in cancer cells: A new strategy for DNA alkylators in cancer therapy

Abstract Background: O6-Methylguanine (O6-MG)-DNA-methyltransferase (MGMT) encodes the DNA-repair protein O6-alkylguanine (O6-AG) DNA alkyltransferase (AGT) that repair DNA adducts caused by alkylating agents. MGMT has important implications in cancer treatment since its expression induces resistance to methylating and chloroethylating anticancer drugs which are effective treatment options in tumours lacking MGMT expression. The demand to find agents that induce MGMT deficiency and increase the susceptibility to alkylating chemotherapeutic agents is therefore highly warranted. Methods: The correlation between Wnt activity and MGMT expression was investigated in primary medulloblastomas, gliomas and colon cancer using gene expression cohorts. Cell lines from medulloblastomas, gliomas, colon cancer and neuroblastomas were examined for Wnt/β-catenin activity and MGMT expression using immunoblotting and Real-Time quantitative PCR (Q-PCR). Small interfering RNA (siRNA) against β-catenin, forced overexpression of β-catenin, and MGMT promotor reporter plasmids were used to study β-catenin mediated regulation on MGMT. Cell cytotoxicity and clonogenicity of chemotherapeutic drugs in combination with celecoxib were examined in cell lines using fluorometric microculture cytotoxicity assay and clonogenic assay, respectively. In vivo, nude NMRI mice carrying D283 MED xenografts were treated with celecoxib in combination with temozolomide. Results: Wnt signaling activity was shown to be correlated to increased MGMT expression levels both in primary tumors and cell lines of different origins. Transfection experiments revealed that β-catenin regulates MGMT expression. Proinflammatory prostaglandin E2 activates the Wnt/β-catenin cascade and increase MGMT expression. The clinically available COX-2 inhibitor celecoxib augmented the cytotoxic effect of the DNA alkylating drug, temozolomide in cells with elevated MGMT expression both in vitro and in established medulloblastoma xenografts in nude mice through inhibition of Wnt/β-catenin activity and subsequent reduction in MGMT expression. Conclusions: This study demonstrates that celecoxib reduces the transcription of the MGMT gene through a prostaglandin E2-Wnt/β-catenin route and thus increases the susceptibility to temozolomide. These finding are the first reported that Wnt/β-catenin pathway regulates the expression of MGMT and thus provides a rational approach for improved efficacy of chemotherapeutic drugs inducing DNA alkylation in cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-209. doi:10.1158/1538-7445.AM2011-LB-209

Sonic Hedgehog Pathway Is Essential for Maintenance of Cancer Stem-Like Cells in Human Gastric Cancer

Abnormal activation of the Sonic hedgehog (SHH) pathway has been described in a wide variety of human cancers and in cancer stem cells (CSCs), however, the role of SHH pathway in gastric CSCs has not been reported. In this study, we investigated the possibility that abnormal activation of the SHH pathway maintained the characteristics of gastric CSCs. First, we identified cancer stem-like cells (CSLCs) from human gastric cancer cell lines (HGC-27, MGC-803 and MKN-45) using tumorsphere culture. Compared with adherent cells, the floating tumorsphere cells had more self-renewing capacity and chemoresistance. The cells expressing CSCs markers (CD44, CD24 and CD133) were also significantly more in tumorsphere cells than in adherent cells. More importantly, in vivo xenograft studies showed that tumors could be generated with 2×104 tumorsphere cells, which was 100-fold less than those required for tumors seeding by adherent cells. Next, RT-PCR and Western blot showed that the expression levels of Ptch and Gli1 (SHH pathway target genes) were significantly higher in tumorsphere cells than in adherent cells. The results of quantitative real-time PCR were similar to those of RT-PCR and Western blot. Further analysis revealed that SHH pathway blocked by cyclopamine or 5E1 caused a higher reduction in self-renewing capacity of HGC-27 tumorsphere cells than that of adherent cells. We also found that SHH pathway blocking strongly enhanced the efficacy of chemotherapeutic drugs in HGC-27 tumorsphere cells in vitro and in vivo but had no significant effect in adherent cells. Finally, we isolated the tumorspheres from gastric cancer specimen, these cells also had chemoresistance and tumorigenic capacity, and SHH pathway maintained the gastric CSLCs characteristics of tumorsphere cells from primary tumor samples. In conclusion, our data suggested that SHH pathway was essential for maintenance of CSLCs in human gastric cancer.

Penetration of paclitaxel and 5-fluorouracil in multicellular layers of human colorectal cancer cells

Limited efficacy of chemotherapeutic drugs against solid tumors has been attributed to poor drug penetration into tumor tissues. Multicellular layer (MCL) cultures recapitulate barriers to drug penetration and distribution and have been used successfully in the production of clinically relevant data. In the present study, we evaluated the characteristics of paclitaxel (PTX) and 5-fluorouracil (5-FU) penetration and their effects on tissue penetration using MCLs of human colorectal cancer cells (DLD-1 and HT-29) grown in Transwell inserts. Drug concentration in conditioned media after MCL penetration was estimated using % survival of cells exposed to the conditioned media, and the penetration rate was calculated as % drug concentration relative to the expected concentration after penetration of cell-free MCLs. PTX showed limited penetration in both MCLs in contrast to the full penetration seen by 5-FU. The penetration rate measured after 24 h by cytotoxicity of the conditioned media was 40 and 38% in DLD-1 (20 μM) and HT-29 MCLs (1 μM), respectively, at which concentration the conditioned media produced 50% growth inhibition in monolayers. The penetration profile obtained using [14C]-paclitaxel also showed slow and limited penetration with concentration- and cell line-dependency. In HT-29 MCL, full penetration of PTX was obtained at 10 μM after 48 h, whereas only 80% was obtained at 1 μM. In DLD-1 MCLs, penetration of PTX was minimal, especially at 1 μM, showing penetration rates as low as 10 and 20% after 24 and 96 h, respectively. When PTX and 5-FU were allowed to penetrate in sequential combination, no effect on the penetration rate was observed. Overall, our results demonstrated limited penetration of PTX in human colorectal cancer MCLs along with concentration-, time-, and cell line-dependency. Assessment of penetration using cytotoxicity of the conditioned media used in the present study may be useful in early stage screening of anticancer agents for their potential in tissue penetration.

Brusatol enhances the efficacy of chemotherapy by inhibiting the Nrf2-mediated defense mechanism

The major obstacle in cancer treatment is the resistance of cancer cells to therapies. Nrf2 is a transcription factor that regulates a cellular defense response and is ubiquitously expressed at low basal levels in normal tissues due to Keap1-dependent ubiquitination and proteasomal degradation. Recently, Nrf2 has emerged as an important contributor to chemoresistance. High constitutive expression of Nrf2 was found in many types of cancers, creating an environment conducive for cancer cell survival. Here, we report the identification of brusatol as a unique inhibitor of the Nrf2 pathway that sensitizes a broad spectrum of cancer cells and A549 xenografts to cisplatin and other chemotherapeutic drugs. Mechanistically, brusatol selectively reduces the protein level of Nrf2 through enhanced ubiquitination and degradation of Nrf2. Consequently, expression of Nrf2-downstream genes is reduced and the Nrf2-dependent protective response is suppressed. In A549 xenografts, brusatol and cisplatin cotreatment induced apoptosis, reduced cell proliferation, and inhibited tumor growth more substantially when compared with cisplatin treatment alone. Additionally, A549-K xenografts, in which Nrf2 is expressed at very low levels due to ectopic expression of Keap1, do not respond to brusatol treatment, demonstrating that brusatol-mediated sensitization to cisplatin is Nrf2 dependent. Moreover, a decrease in drug detoxification and impairment in drug removal may be the primary mechanisms by which brusatol enhances the efficacy of chemotherapeutic drugs. Taken together, these results clearly demonstrate the effectiveness of using brusatol to combat chemoresistance and suggest that brusatol can be developed into an adjuvant chemotherapeutic drug.

In Vitro and In Vivo Inhibition of MRP Gene Expression and Reversal of Multidrug Resistance by siRNA

Clinical drug resistance to chemotherapeutic agents is one of the major hindrances in the treatment of human cancers. One mechanism by which a living cell can achieve multidrug resistance (MDR) is via the active efflux of a broad range of anti-cancer drugs through the cellular membrane by MDR proteins. Over-expression of multidrug resistance-associated protein 1 (MRP1) is one of the important MDR phenotypes. RNA interference (RNAi) is a fundamental cellular mechanism for silencing gene expression that can be harnessed for the development of new drugs. In our study, by using lipofectamin(TM) 2000 (Invitrogen, Carlsbad, CA, USA) in vitro and electric pulse in vivo to delivery siRNA, we successfully inhibited MRP1 both at mRNA and protein level as determined by reverse transcription-PCR and western blot or immunohistochemistry. Furthermore, the efficacy of chemotherapeutic drugs (epirubicin) to tumour cells dramatically improved both in vivo and in vitro. These studies demonstrate that through efficient delivery siRNA, MRP1-mediated MDR can be reversed and siRNA can be used for further study in clinical cancer therapy.

Image-Based Chemical Screening Identifies Drug Efflux Inhibitors in Lung Cancer Cells

Cancer cells with active drug efflux capability are multidrug resistant and pose a significant obstacle for the efficacy of chemotherapy. Moreover, recent evidence suggests that high drug efflux cancer cells (HDECC) may be selectively enriched with stem-like cancer cells, which are believed to be the cause for tumor initiation and recurrence. There is a great need for therapeutic reagents that are capable of eliminating HDECCs. We developed an image-based high-content screening (HCS) system to specifically identify and analyze the HDECC population in lung cancer cells. Using the system, we screened 1,280 pharmacologically active compounds that identified 12 potent HDECC inhibitors. It is shown that these inhibitors are able to overcome multidrug resistance (MDR) and sensitize HDECCs to chemotherapeutic drugs, or directly reduce the tumorigenicity of lung cancer cells possibly by affecting stem-like cancer cells. The HCS system we established provides a new approach for identifying therapeutic reagents overcoming MDR. The compounds identified by the screening may potentially be used as potential adjuvant to improve the efficacy of chemotherapeutic drugs.

The impact of Akt and nuclear factor kB pathway on chemoresistance of gastric cancer cell

Objective To evaluate the role of Akt and nuclear factor （NF）-kB pathway in the development of chemoresistance in gastric cancer and the relation between Akt and NF-kB. Methods SGC-7901 cells were exposed to chemotherapeutic drugs （ doxorubicin and etoposide ） or chemotherapeutic drugs combined with Wortmannin or MCr132. The cell growth was detected using MTT method. The apoptosis of SGC-7901 cells was measured by TUNEL and Annexin V/PI methods. The protein level of NF-kB was analyzed by immunocytochemical staining. Electrophoretic mobility shift assay （EMSA） was used to confirm the increased nuclear translocation of NF-kB/P65. The protein levels of p-Akt and p-IkBa were determined by Western blotting. Results① Both chemotherapeutic drugs could obviously inhibit the growth of SGC-7901 cells in time-dose-dependent manner. Pretreatment of SGC-7901 cells with Wortmannin or MG-132 could promote this inhibitory effect. ② Both chemotherapeutic drugs could induce apoptosis of SGC-7901 and activate Akt and NF-kB in a close-dependent manner. Wortmannin or MG-132 pretreatment could enhance the apoptosis of SGC 7901 cells and bloke NF-kB activation induce by doxorubicin or etoposide.③ The activation of NF-kB was found in SGC-7901 cells stimulated with Wortmannin,but no activation of Akt was noted in those treated with MG-132. Conclusions The chemotherapeutic drugs can both induce apoptosis and activate Akt and NF-kB in SGC-7901 cells. The efficacy of chemotherapeutic drugs can be increased via inhibiting activation of Akt or NF-kB. Key words: Stomach neoplasms; Chemotherapy; Drug resistance

High Levels of Nrf2 Determine Chemoresistance in Type II Endometrial Cancer

Type II endometrial cancer, which mainly presents as serous and clear cell types, has proved to be the most malignant and recurrent carcinoma among various female genital malignancies. The transcription factor Nrf2 was first described as having chemopreventive activity. Activation of the Nrf2-mediated cellular defense response protects cells against the toxic and carcinogenic effects of environmental insults by upregulating an array of genes that detoxify reactive oxygen species and restore cellular redox homeostasis. However, the cancer-promoting role of Nrf2 has recently been revealed. Nrf2 is constitutively upregulated in several types of human cancer tissues and cancer cell lines. Furthermore, inhibition of Nrf2 expression sensitizes cancer cells to chemotherapeutic drugs. In this study, the constitutive level of Nrf2 was compared in different types of human endometrial tumors. It was found that Nrf2 was highly expressed in endometrial serous carcinoma (ESC), whereas complex hyperplasia and endometrial endometrioid carcinoma (EEC) had no or marginal expression of Nrf2. Likewise, the ESC-derived SPEC-2 cell line had a higher level of Nrf2 expression and was more resistant to the toxic effects of cisplatin and paclitaxel than the Ishikawa cell line, which was generated from EEC. Silencing of Nrf2 rendered SPEC-2 cells more susceptible to chemotherapeutic drugs, whereas it had a limited effect on Ishikawa cells. Inhibition of Nrf2 expression by overexpressing Keap1 sensitized SPEC-2 cells or SPEC-2-derived xenografts to chemotherapeutic treatments using both cell culture and severe combined immunodeficient mouse models. Collectively, we provide a molecular basis for the use of Nrf2 inhibitors to increase the efficacy of chemotherapeutic drugs and to combat chemoresistance, the biggest obstacle in chemotherapy.

Inhibition of metalloproteinases derived from tumours: new insights in the treatment of human glioblastoma

Glioblastoma multiforme is the most commonly diagnosed malignant primary brain tumour in adults. Invasive behaviour is the pathological hallmark of malignant gliomas; consequently, its inhibition has been suggested as a therapeutic strategy. Tumour cell-derived gelatinases (matrix metalloproteinase-2, matrix metalloproteinase-9) can be considered prime factors in glioma invasiveness: their expression correlates with the progression and the degree of malignancy. Thus, broad spectrum matrix metalloproteinase inhibitors (MMP inhibitors) have been included in clinical trials. In the present study, the invasiveness, viability and progression of the human glioma cell line U87MG were investigated following treatment with N-O-isopropyl sulfonamido-based hydroxamates (compounds 1 and 2) as MMP-2 inhibitors used at nanomolar concentration. A standard broad spectrum MMP-inhibitor belonging to the classical tertiary sulfonamido-based hydroxamates family (CGS_27023A) was used too. The compounds 1 and 2 resulted in potent inhibition of cell invasiveness ( P<0.0001) without affecting viability. In some clinical trials, the combined therapy of temozolomide (an alkylating agent used in glioma treatment) plus marimastat (a broad spectrum MMP inhibitor) has provided evidence of the importance of MMPs to tumor progression and invasiveness. On this basis, the effect on U87MG cells of a combined treatment with temozolomide, plus each of the two MMP inhibitors at nanomolar concentration, was investigated. The obtained data demonstrated the inhibition of cell invasiveness and viability after treatment. These results can help in developing clinical combined therapy using MMP inhibitors that, at low doses, increase the anticancer efficacy of chemotherapeutic drugs, probably without causing the side effects typical of broad-spectrum MMP inhibitors.

Effect of Combination Treatment of Rapamycin and Isoflavones on mTOR Pathway in Human Glioblastoma (U87) Cells

Glioblastoma Multiforme (GBM) is a malignant primary brain tumor associated with poor survival rate. PI3K/Akt pathway is highly upregulated in gliomas due to deletion or mutation of PTEN and its activation is associated with tumor grade. mTOR is downstream from PI3K/Akt pathway and it initiates translation through its action on S6K and 4E-BP1. mTOR is an important therapeutic target in many cancers, including glioblastomas. Rapamycin and its analogues are known to inhibit mTOR pathway; however, they also show simultaneous upregulation of Akt and eIF4E survival pathways on inhibition of mTOR, rendering cells more resistant to rapamycin treatment. In this study we investigated the effect of combination treatment of rapamycin with isoflavones such as genistein and biochanin A on mTOR pathway and activation of Akt and eIF4E in human glioblastoma (U87) cells. Our results show that combination treatment of rapamycin with isoflavones, especially biochanin A at 50 muM, decreased the phosphorylation of Akt and eIF4E proteins and rendered U87 cells more sensitive to rapamycin treatment when compared to cells treated with rapamycin alone. These results suggest the importance of combining chemopreventive with chemotherapeutic agents in order to increase the efficacy of chemotherapeutic drugs.

Rationale for the Use of Natural Anti-Inflammatory Agents in Cancer Chemotherapy

Chemotherapeutic drugs have been conventionally used for the cancer treatments, but the efficacy of the therapy remains problematic. It has been strongly suggested that the anti-inflammatory drugs, such as COX-2 inhibitors, may enhance the anti-cancer efficacy of chemotherapeutic drugs and reduce the chemoresistance. Herein, the rational of the use of antiinflammatory agents in the cancer treatment is summarized. One of the most important inflammatory processes occurs during metabolism of the eicosanoids, which involved arachidonic acid (AA), cyclooxygenase 2 (COX-2), prostaglandins (PGs) and leukotriens (LTs). As the results of excessive up-regulation of COX-2 and production of PGE2, for example, the harmful inflammation may promote the cancer development. The inflammation also involve in the drug resistance of the chemotherapy. In fact, many in vitro and in vivo studies have showed the positive potentials using COX-2 inhibitors in the cancer treatment. However, the regular anti-inflammatory agents, mainly COX-2 inhibitors, may