NSC23925 And CD44 Targeted HA-PEI/HA-PEG/MDR1 SiRNA Nanoparticle Circumvent Drug Resistance In Ovarian Cancer

BackgroundOvarian cancer ranks as the leading cause of gynecological cancer deaths among women. Three-quarters of patients diagnosed with ovarian cancer present with advanced disease as they are typically asymptomatic at the early stage. Primary surgical cyto-reduction followed by the combination of paclitaxel and platinum chemotherapy is the recommended standard treatment for advanced ovarian cancer. Unfortunately, more than half of advanced ovarian cancer patients will ultimately develop recurrence, and become resistant to paclitaxel, as well as cross-resistant to other chemotherapeutic drugs. One of the best characterized multidrug resistance(MDR) mechanisms is overexpression of multidrug resistance gene 1(MDR1) and corresponding P-glycoprotein(Pgp). Overexpression of MDR1/Pgp has been found in multiple MDR ovarian cancer cell lines and tissues. The development of MDR severely limits the success of chemotherapy in ovarian cancer treatment. Circumventing drug resistance has been a high priority of clinical and investigational oncology, but remains an elusive goal. Preventing the development of drug resistance in sensitive cancer cells and reversing drug reistance in MDR cancer cells have been regarded as the promising strategies for overcoming drug resistance.It has been demonstrated that the emergence of MDR can be prevented using well-characterized Pgp inhibitors valspodar(PSC833), biricodar(VX-710), and tariquidar(XR9576), however, no significant survival benefits of these regaents have been established in clinical trials. Therefore, development of more potent and selective MDR preventers is needed. NSC23925(2-(4-methoxyphenyl)-4- quinolinyl)(2-piper-idinyl) methanol) is a small molecular compound which was identified as an effective and selective inhibitor of Pgp in our previous studies. NSC23925 has been proven to have the ability to reverse Pgp-mediated MDR by stimulating Pgp ATPase activity in MDR ovarian cancer in vitro and in vivo. However, whether NSC23925 can prevent the emergence of MDR in ovarian cancer during continued paclitaxel treatment is yet to be determined.MDR1 small interfering RNA(si RNA) based strategies which can decrease the expression levels of MDR1 and Pgp were also proposed to circumvent MDR, however, systemic, safe, and effective targeted delivery is still a major challenge. Recently, nanoparticles have been shown to be one of the promising si RNA delivery vehicles based on their unique physical and biological properties. Low molecular weight hyaluronic acid(HA) polymer was used as our starting building block due to their biodegradability, nonimmunogenicity, non-toxic profile and easily modification. Moreover, HA has a high affinity and specificity for cluster of differentiation 44(CD44) receptors, which higly expresses in drug resistant ovarian cancer cells. Previously, CD44 targeted HA based nanoparticle has been shown to successfully deliver chemotherapy agents or si RNA into tumor cells. However, the effects of HA-poly(ethyleneimine)/HA-poly(ethylene glycol)(HA-PEI/HA-PEG) nanoparticle on delivery MDR1 si RNA and reversing drug resistance in ovarian cancer are unkown. Therefore, the above issues were elucidated in 3 different parts in the current thesis. Part Ⅰ NSC23925 prevents the emergence of multidrug resistancein ovarian cancer in vitro ObjectivesTo evaluate the effects of NSC23925 on prevention of the development of MDR in ovarian cancer during continued paclitaxel treatment, and elucidate the underlying mechanisms. Materials and methodsTo establish MDR ovarian cancer cell sublines, human ovarian cancer cells, OVCAR8 and SKOV-3, were cultured with either increasing concentrations of paclitaxel alone or in combination with NSC23925(palcitaxel/NSC23925). Cell sublines selected at different selection points were evaluated for their drug sensitivity, MDR1 expression, and Pgp expression and activity by MTT, Real-time PCR, Western blot, immunofluorescene(IF) and drug uptake/efflux assay. Results1. NSC23925 prevented the development of MDR in ovarian cancer cells during continued paclitaxel treatment.(1) OVCAR8 and SKOV3 cells selected with stepwise increased concentration of paclitaxel alone showed stable growth in culture medium supplemented with 0.3 μM paclitaxel(labeled with OVCAR8/paclitaxel0.3 and SKOV-3/paclitaxel0.3, respectively). In sharp contrast, OVCAR8 and SKOV3 cells cultured with palcitaxel/NSC23925 could only grow stably in the presence of 0.006 μM and 0.001 μM paclitaxel, respectively(labeled with OVCAR8/paclitaxel0.006-NSC23925 and SKOV-3/paclitaxel0.001-NSC23925, respectively).(2) OVCAR8/paclitaxel0.3 and SKOV-3/paclitaxel0.3 cells displayed significantly higher IC50 of paclitaxel as compared with parental cells, while no obvious increase in the IC50 of paclitaxel was observed in OVCAR8/paclitaxel0.006-NSC23925 and SKOV-3/paclitaxel0.001-NSC23925 cells.(3) OVCAR8/paclitaxel0.3 cells appeared cross-resistance to doxorubicin, vincristine, and docetaxel, while OVCAR8/paclitaxel0.006-NSC23925 cells remained sensitive to above drugs.2. NSC23925 prevented the development of MDR by inhibiting the overexpression of Pgp and MDR1 in ovarian cancer cells. Pgp was significantly overexpressed in OVCAR8/paclitaxel0.3 and SKOV-3/paclitaxel0.3 cells compared with OVCAR8/paclitaxel0.006-NSC23925 and SKOV-3/paclitaxel0.001-NSC23925 cells. Moreover, a dramatic enhancement of MDR1 was detected in OVCAR8/paclitaxel0.3 cells compared to OVCAR8/paclitaxel0.006-NSC23925 cells.3. NSC23925 maintained intracellular retention of chemotherapeutic agents, and subsequently remained cell sensitivity to chemotherapy during continued paclitaxel treatment in ovarian cancer cells. Compared with OVCAR8/paclitaxel0.3 cells, a statistically higher intracellular accumulation of Calcein AM, doxorubicin, Rhodamine(R123) and Dioc 2, and a considerably lower relative efflux level of R123 were exhibited in OVCAR8/paclitaxel0.006-NSC23925 cells.4. No obvious changes in the sensitivity to chemotherapeutic agents, and Pgp expression and activity were detected in ovarian cancer cells grown with NSC23925 alone. Brief summaryThe emergence of MDR could be prevented by NSC23925 through inhibiting the overexpression of Pgp and MDR1, and subsequently influencing the function of Pgp in ovarian cancer during continued paclitaxel treatment in vitro.Part Ⅱ NSC23925 prevents the development of paclitaxel resistancein ovarian cancer in vivo ObjectivesThe goal of our current study was to determine whether the combination of paclitaxel and NSC23925 can prevent the development of paclitaxel resistance in ovarian cancer xenograft mouse model in vivo, and to further elucidate the underlying molecular mechanisms. Materials and methodsAanimal studies were carried out with protocol(#2013N000121) approved by the Massachusetts General Hospital Subcommittee on Research Animal Care(SRAC). To establish drug sensitive ovarian cancer mouse model, approximately 2 × 106 parental sensitive SKOV-3 cells were injected subcutaneously with Matrigel into the flanks of 3- 4-week-old female nude mice. The paclitaxel-resistant ovarian cancer mouse models were then established by continuous paclitaxel treatment in combination with or without NSC23925 administration in the mice bearing subcutaneous SKOV-3 xenografts. The health of the mice was monitored daily, and the size of tumors and the weight of mice were measured twice per week throughout the experiment. Mice were sacrificed at the end of the experiments, and tumor tissues, peripheral blood, liver, kidney, and spleen were collected. The expression levels of Pgp and several apoptotic-related proteins were evaluated in mouse tumor samples by Western blot. Safety evaluation following the treatment with paclitaxel/NSC23925 combination in tumor bearing mice was carried out by assessing the changes in body weight, white blood cell(WBC) and red blood cell(RBC) count, and the liver, kidney, spleen histology. Results1. The optimal concentration of paclitaxel in mice for the establishment of paclitaxel-resistant cells was 25 mg/kg.2. NSC23925 in combination with paclitaxel had the ability to prevent the emergence of paclitaxel resistance by specifically preventing the overexpression of Pgp in ovarian cancer mice model.(1) Consistent with clinical observation, paclitaxel treated tumors eventually experienced an increase in tumor volume after initial stability in tumor size during the continued paclitaxel treatment, whereas no obvious tumor growth was detected in paclitaxel-NSC23925 treated tumors during the continued treatment. Paclitaxel-NSC23925 treated mice showed marked and sustained response to paclitaxel without becoming paclitaxel-refractory.(2) A majority of paclitaxel treated mice showed significant increase in Pgp expression level. In contrast, the expression level of Pgp was undetectable in all paclitaxel-NSC23925 treated mice. Paclitaxel-NSC23925 treated mice revealed the remarkable lower Pgp level as compared to paclitaxel treated mice(P < 0.01).3. The combination of paclitaxel and NSC23925 prompted the apoptosis in mouse models. Anti-apoptotic proteins survivin, Bcl-x L, and MCL-1 showed significantly lower expression levels in paclitaxel-NSC23925 treated mice than paclitaxel alone treated mice(P < 0.001, P < 0.05 and P < 0.01, respectively). CD44 and Integrin β3 also displayed significantly decreased expression in paclitaxel-NSC23925 treated mice as compared with that in paclitaxel alone treated mice(P < 0.05 and P < 0.01, respectively).4. No remarkable toxicities were observed when NSC23925 was administered either alone or in combination with paclitaxel over a long period of time. There were no significant differences in the body weight, RBC and WBC number between paclitaxel treated and paclitaxel-NSC23925 treated mice(P > 0.05). In addition, no apparent pathological changes were detected in liver, kidney, and spleen among different groups. Brief summaryPaclitaxel-NSC23925 combinations can prevent the development of paclitaxel resistance in ovarian cancer in vivo, which is due to its function specifically inhibiting the overexpression of Pgp, as well as blocking the expression of anti-apoptotic proteins.Part Ⅲ CD44 targeted HA-based nanoparticles for MDR1 si RNA delivery to reverse drug resistance in ovarian cancer ObjectivesThe aim of this study was to evaluate the ability of HA-PEI/HAI-PEG to deliver MDR1 si RNA and the efficacy of the combination of HA-PEI/HA-PEG/MDR1 si RNA with paclitaxel to reverse drug resistance in MDR ovarian cancer both in vivo and in vitro. Materials and methodsHA-PEI/HA-PEG nanoparticles were synthesized and characterized, then the cellular uptake and knockdown efficiency of HA-PEI/HA-PEG/MDR1 si RNA nanoparticles was further determined. A human xenograft MDR ovarian cancer model was established to evaluate the effects of the combination of HA-PEI/HAPEG/MDR1 si RNA nanoparticles and paclitaxel on MDR tumor growth, Pgp expression and apoptosis. Results1. CD44 was highly expressed in drug resistant cells, OVCAR8 TR and SKOV-3TR, and in human ovarian cancer tissues derived from patients.2. The Z-average particle size and zeta potential of HA-PEI/HA-PEG/MDR1 si RNA nanoparticles was 173.3 ± 13.7 nm and-22.5 ± 0.44 m V, respectively. Moreover, ovarian cacner cells could tolerate HA-PEI/HA-PEG/MDR1 si RNA well during transfection.3. HA-PEI/HA-PEG nanoparticles successfully delivered MDR1 si RNA into ovarian cancer cells. Moreover, HA-PEI/HA-PEG nanoparticles loaded with MDR1 si RNA efficiently down-regulated the expression of MDR1 and Pgp, inhibited the functional activity of Pgp, and subsequently increased cell sensitivity to paclitaxel in cultured MDR ovrain cancer cells.4. Adminstration of HA-PEI/HA-PEG/MDR1 si RNA nanoparticles followed by paclitaxel treatment induced a significant inhibitory effect on the tumor growth, decreased Pgp expression and increased apoptosis in MDR ovarian cancer mice model. Brief summaryCD44 targeted HA-PEI/HA-PEG nanoparticles can successfully delivery MDR1 si RNA into ovarian cancer. HA-PEI/HA-PEG/MDR1 si RNA nanoparticles have the abilities to decrease expression levels of MDR1 and Pgp, increase apoptosis and therefore reverse drug resistance in ovarian cancer in vitro and in vivo.Conclusions1. Paclitaxel-NSC23925 combination can prevent acquired drug resistance in ovarian cancer cells in vitro and in vivo. The clinical use of NSC2395 at the onset of chemotherapy may prevent the development of Pgp-mediated drug resistance and improve the clinical outcome of patients with ovarian cancer.2. CD44 targeted HA-PEI/HA-PEG/MDR1 si RNA nanoparticles can serve as a therapeutic tool with great potentials to reverse drug resistance, and improve the clinical outcome in ovarian cancer.