Leptin exerts its functional effects through bindingto the leptin receptors, OB-Rb and OB-Ra expressed in ovariancancer cells (). In the presentstudy, we examined the effect of leptin on ovarian cancer cellgrowth using trypan blue exclusion staining and MTT assay. OVCAR-3cells were serum-deprived for 48 h and then treated with variousconcentrations of leptin for 48 h. MTT assay showed that leptinstimulated OVCAR-3 cell growth in a dose-dependent manner. Thestimulation was observed at a dose as low as 50 ng/ml (). Using the trypan blue exclusionassay to estimate the amount of viable cells, the results showedthat leptin enhanced cell growth not only in a dose-dependent butalso in a time-dependent manner ().
Up to date, there have been many published researchstudies about the correlation of miRNAs with EOC. Lu reported that hypermethylation of let-7a-3 in epithelial ovariancancer was associated with low insulin-like growth factor-IIexpression and favorable prognosis (). Additionally, the beneficial impactof the addition of paclitaxel on EOC survival has beensignificantly linked to let-7a levels, and it has been shown thatmiRNAs such as let-7a may be useful markers for the selection ofchemotherapeutic agents in EOC management (). Lou reported thatmicroRNA-21 could promote the cell proliferation, invasion andmigration abilities in ovarian epithelial carcinomas throughinhibiting the expression of PTEN protein (). In other studies, several othermicroRNAs have been reported to be associated with chemotherapyresistance, such as miR-214, miR-130a, miR-27a and miR-451(–). Although miR-100 was reported to bedownregulated in human EOC by other research groups, theclinicopathological or prognostic significance of miR-100 in EOC isstill unknown. In nasopharyngeal cancer, underexpressed miR-100 wasfound to lead to PLK1 overexpression, which in turn contributes toNPC progression (). However, inprostate cancer, it was reported that a high level of miR-100 wasrelated to biochemical recurrence of localized prostate cancer inpatients treated with radical prostatectomy (). Recently, Zheng and his groupreported that miR-100 could regulate cell differentiation andsurvival by targeting RBSP3, a phosphatase-like tumor suppressor inacute myeloid leukemia (). Fromthe above studies, it may be concluded that miRNA oncogenes andtumor suppressors show different patterns in different tumortypes.
After confirming that HA-PEI/HA-PEG CD44 targeted nanoparticles can efficiently deliver MDR1 siRNA into MDR cells and result in down-regulation of MDR1 and Pgp expression, we next evaluated antitumor efficacy of the combination of HA-PEI/HA-PEG/MDR1 siRNA CD44 targeted nanoparticles and paclitaxel in ovarian cancer tumor-bearing nude mice. The treatment scheme is shown in . Our in vivo study showed that the average tumor volume in mice administrated with the combination of HA-PEI/HA-PEG/MDR1 siRNA CD44 targeted nanoparticle and paclitaxel was significantly smaller than that observed in control groups (). Tumor volume of mice treated with paclitaxel alone was approximately 3-fold higher than in mice treated with combined HA-PEI/HA-PEG/MDR1 siRNA CD44 targeted nanopartricle and paclitaxel, which could reflect an increased chemosensitivity to paclitaxel in mice treated with HA-PEI/HA-PEG/MDR1 siRNA. However, changes in tumor volume in mice treated with either the combination of HA-PEI/HA-PEG/non-specific siRNA CD44 targeted nanoparticle and paclitaxel or the combined naked MDR1 siRNA and paclitaxel was similar to that with paclitaxel treated alone in this ovarian cancer MDR model (). Representative images of mice and tumors form each group were shown in .
Lu L, Katsaros D, de la Longrais IA,Sochirca O and Yu H: Hypermethylation of let-7a-3 in epithelialovarian cancer is associated with low insulin-like growth factor-IIexpression and favorable prognosis. Cancer Res. 67:10117–10122.2007. : :
Several studies have demonstrated that natural compounds-induced cell growth inhibition in cancer cells could be related with increase of DR pathway, an important extrinsic apoptotic pathway. Indomethacin and sulindac sulfide, one of the major metabolites of sulindac, activate caspase 8 and induce apoptosis by a fas-associating protein with death domain (FADD)-dependent mechanism in Jurkat T cells . Sulindac sulfide is also believed to mediate its antitumorigenic effects by inducing apoptosis through up-regulated DR5 and activated the caspase 8 in colon and prostate cancer cell lines . Our previous study also showed that BV and Snake venom toxin induced apoptotic cell death of colon, prostate, and ovarian cancer cells through enhancement of DRs (DR3, DR4, DR5 and DR6) expression. Our present results showed that expression of DR proteins such as DR3 and DR6 in Ca Ski and C33A cervical cancer cell were increased. However, treatment of DR3 and DR6 siRNA in Ca Ski and C33A reversed BV-induced cervical cancer cell growth inhibition. We also found that DR3 and DR6 expression was significantly higher in the BV treated cultured human cervical cancer cells as well as cervical tumor tissues and xenograft tumor tissues. Selective triggering of DR expression is implicated in the induction of cancer cell death and the expression of DR is varying depending on cell types and stimuli conditions. These data indicated that higher DR3 and DR6 expression could be significant for anti-cancer effect of BV.
A number of studies have revealed that the highlytumorigenic cancer progenitor cells expressing stem cell-likemarkers, such as CD133, CD44, Oct-3/4, c-KIT and/or xenobioticefflux pumps associated with multidrug resistance, have beenisolated from ovarian cancers and tumors established with cancercell lines (–). These cancer progenitor cells havealso been designated as cancer stem cells or cancer-initiatingcells, which are able to give rise to more differentiated cancercell types and and appear to play acritical role in tumor formation, progression and metastasis. Tofurther demonstrate the characteristics of TAFs and NFCs, weexamined their stem cell gene expression. We found that theexpression of stem cell genes was higher in TAFs than that innormal fibroblasts. A number of studies have demonstrated thatnormal fibroblasts play a role in maintaining epithelialhomeostasis by suppressing the proliferation and oncogenicpotential of adjacent epithelia (,).The cellular origin of TAFs remains unclear; however, following theneoplastic transformation of epithelia, some TAFs are recruited tothe expanding tumor mass from local tissue fibroblasts () and additional TAFs can be recruitedfrom peripheral fibroblast pools, such as bone marrow-derivedmesenchymal stem cells (MSCs) (). Spaeth providedevidence that TAFs are derived from MSCs that acquire a TAFphenotype following exposure to or systemic recruitment into axenograft ovarian adenocarcinoma model (). In this manner, the upregulatedexpression of diverse tumorigenic target gene products in cancerprogenitor cells and their progeny induced through the activationof distinct developmental signaling pathways, such as EGF/epidermalgrowth factor receptor (EGFR), hedgehog, Wnt/β-catenin, Notch,tumor growth factor-β (TGF-β) and/or integrin cascades mayco-operatively participate in the formation of TAFs (). Thus, the acquisition of stem cellcharacteristics of the fibroblasts may be determined by theirsource. Recently published data suggest that TAF-induced EMT leadsto the enhanced expression of stem cell markers in prostatecarcinoma cells, and increases the ability of these cells to formprostaspheres and to self-renew (). Thus, the paracrine interplaybetween TAFs and cancer cells leads to the maintaining of cancerstem cell properties associated with aggressiveness and metastaticspread.
The expression of human FAP is highly specific fortumor fibroblasts. FAP is heavily expressed in reactive stromalfibroblasts in >90% of human epithelial carcinomas includingbreast, lung, colorectal and ovarian. Neuronal and lymphoid cells,as well as the surrounding normal tissue, demonstrate a very weakFAP expression (). Cheng reported that HEK293 cells ectopically overexpressing FAP,when xenografted into scid mice, were 2–4-fold more likely todevelop tumors and showed that FAP increased tumorigenicity andsignificantly enhanced tumor growth (). They also found that enzymaticmutants of FAP that are devoid of FAP enzymatic activity, whenxenografted into immunodeficient mice, resulted in attenuated tumorgrowth (). In the presentstudy, we found that silencing FAP inhibited the growth of TAFs,accompanied with cell cycle arrest at the G2 and S phase. Of note,FAP siRNA transfection also significantly inhibited the stem cellgene expression in TAFs.
In the current study, we investigated whether leptincan stimulate ovarian cancer cell growth and prevent apoptosisunder serum-starvation conditions. We first observed that leptinstimulated the expression of the anti-apoptotic protein, Mcl-1. Ourdata demonstrate that leptin enhances cell growth by activating theJAK2, MEK/ERK1/2 and PI3K/Akt pathways in ovarian cancer. Ourresults indicate that leptin plays an important role betweenobesity and the progression of ovarian cancer.
In conclusion, we show that TAFs have thecharacteristics of stem cells and that FAP silencing in TAFsinhibits cell growth as well as stem cell geneexpression. In addition, we show that FAP silencing in SKOV3 cellsinduces ovarian tumors, but significantly reduces tumor growth inthe xenograft mouse model, accompanied with the decrease of TAFcharacteristics. Clearly, FAP plays an important role in thetumorigeness, stromagenesis and angiogenesis of ovarian cancer andtargeting FAP is a potential therapeutic strategy for ovariancancer patients.
Cyclin D1 is a growth sensor induced by a variety ofgrowth factors and mitogens to trigger cell cycle progression(). Therefore, we used westernblot analysis to measure the expression of cyclin D1 in OVCAR-3cells. At a low concentration (50 ng/ml), leptin increased theexpression of cyclin D1 and the effect of leptin on cyclin D1expression reached a maximum at the concentration of 200 ng/ml(). Since leptin exertsanti-apoptotic effects on OVCAR-3 cells, we sought to determine themechanisms responsible for this response. Usually, theoverexpression of Mcl-1 delays apoptosis induced by growth factorwithdrawal (). Increased Mcl-1expression has been associated with poor prognosis in ovariancancer (). OVCAR-3 cellstreated with leptin showed a dose-dependent increase in the amountof Mcl-1 protein. The effect was maximal at 400 ng/ml leptintreatment (). As shown in, leptin acutely stimulatedMcl-1 expression within 5–60 min; this effect diminished after 2 hof treatment. A second peak in leptin-induced Mcl-1 expression wasdetected at 24 h ().
Activated STAT3 had been shown to directlycontribute to oncogenesis through the upregulation of genesencoding apoptosis inhibitors and cell-cycle regulators, such asBcl-xL, Mcl-1 and cyclin D1/D2, resulting in increased cellproliferation and the prevention of apoptosis in a variety of humancancer cells (). During cancerdevelopment and progression, anti-apoptotic proteins are usuallyoverexpressed and result in the cancer cells becoming resistant toapoptosis (). Mcl-1, a memberof the Bcl-2 family, was first cloned from the human myeloblasticleukemia cell line, ML-1 ().Mcl-1 acts as an anti-apoptotic factor in various tumors, such ashuman myeloid leukemia and hepatocellular carcinoma (). Immunohistochemistry andsemi-quantitative PCR analyses of Mcl-1 expression in ovariancancer patients, have demonstrated that an increased Mcl-1expression is associated with poor prognosis in ovarian cancerpatients. High Mcl-1 expression has been shown to significantlycorrelate with advanced clinical stage, high histopathologicalgrade and poor survival ().Various growth factors and cytokines have been reported to induceMcl-1 expression, such as vascular endothelial growth factor(VEGF), interleukin (IL)-3 and IL-6 (–).