The Function Of CD44 In The Aggressive Progress Of Ovarian Cancer/osteosarcoma And The Study Of Upconversion Nanoparticle In Reversing Tumor Drug Resistance

Background Cancer is the first leading public health issue. High expression of some specific cell surface proteins has been found to contribute to tumor initiation, progression, and poor clinical outcome. Among these proteins, Cluster of Differentiation 44(CD44) is a polymorphic group of proteins, mainly owing to alternatively spliced exon transcripts in the extracellular domains. The CD44 transmembrane glycoprotein family mediates diverse cellular processes, which involve the regulation of growth, CD44 is a receptor for hyaluronic acid(HA) and can interact with other ligands. The binding of different ligands to CD44 triggers direct cross-signaling between different signaling pathways, including HER2, Src kinase, and ERK. Epithelium ovarian carcinoma ranks fifth in lethal tumors among women, accounting for more deaths than any other cancer of the female genital tract. Conventional combinations of primary cytoreductive surgery and paclitaxel-platinum chemotherapy have not had a significant impact on overall survival of ovarian cancer in last several decades. However, the mechanisms of development of relapsed ovarian cancer are still largely unknown. Despite CD44 is frequently expressed in a wide variety of epithelial malignancies, including ovarian cancer. However, the clinical significance of CD44 remains controversial. Few studies have investigated CD44 expression in ovarian cancer patients with long-term follow up. No previous CD44 studies have been carried out with paired primary, metastatic, and recurrent tumor tissues from each individual ovarian cancer patient.Osteosarcoma is an aggressive primary sarcoma of bone. The cellular and molecular mechanisms underlying metastasis formation and the development of chemoresistance in osteosarcoma remain unclear. Mice with disruption of the CD44 gene showed virtually ablated metastasis formation in osteosarcoma. However, few reports have studied the role of CD44 in a larger number of osteosarcoma patients with well-established clinical information. Micro RNAs(mi RNAs, mi Rs) bind to the 3′-untranslated region(3′-UTR) of specific gene m RNAs and participate in the regulation of many biological functions in tumor cells. In addition, we need further identify the specific mi R targeting CD44 in the progression of osteosarcoma.Development of new therapeutic strategies to overcome drug resistance for the treatment of ovarian cancer, osteosarcoma and other cancers is of vital importance. The development of multidrug resistance(MDR) pathways is major obstacle for current cancer chemotherapy. The best known MDR mechanism involves the overexpression of P-glycoprotein(Pgp), the protein product of MDR1. Pgp acts as drug efflux pump the effluxes chemotherapeutic drugs out of the cell, thereby contributes to MDR by keeping intracellular drug concentration at low level. Pgp therefore becomes a promising target to overcome MDR. With the discovery of RNA interference(RNAi) in 1998, small interfering RNA(si RNA) has emerged as promising tool for silencing MDR relevant gene expression, paving the way for the re-sensitizing MDR cancer cells to chemotherapeutic drugs. Effective delivery, tracking and localization of gene during transfection process are of vital importance to understand the intracellular fate of gene. Advance in nanotechnology that provides nanoparticle based multifunctional delivery platform for gene delivery has attracted increasing attention. Most nanoparticle based delivery platform apply UV/short visible light as excitation source for tracking applications, which however suffer from phototoxicityand poor tissue penetration depth resulting in limited clinical potential. Alternatively, upconversion nanoparticles(UCNPs) feature advanced optical properties enabling conversion of near infrared(NIR) excitation to visible emission.Low photo energy and deep tissue penetration of NIR endow UCNPs with many advantages including non-photodamage, non-autofluorescence, non-photobleaching and deep tissue reaching. UCNPs are thus receiving intensive study for gene delivery and tracking. However, despite accumulating work witness the application of UCNPs in gene delivery and tracking, the possibility of UCNPs in gene delivery for the treatment of MDR cancer cells and simultaneously track the intracellular fate of gene have not yet been explored.Section I Up-regulation of CD44 in the development of metastasis, recurrence and drug resistance of ovarian cancerObjective The aim of this study is to evaluate the clinical significance of CD44 expression by using a unique tissue microarray, and then to determine the biological functions of CD44 in ovarian cancer.Methods 1. CD44 expression in tissue microarray(TMA) was assessed by immunohistochemistry. A unique ovarian cancer TMA was constructed with paired primary, metastatic, and recurrent tumor tissues from 26 individual patients in Massachusetts General Hospital. 2. Western blot determined the expression of CD44 in the tumor tissues of a paclitaxel treated ovarian cancer xenograft mouse model. 3. Western blot and immunofluorescenceassay determined the expression of CD44 in ovarian cancer cell lines. 4. MTT assay and 3D culture were used to detect the cell proliferation and the formation of tumor spheroid. 5. MTT assay was applied to asssess the paclitaxel sensitivity. 6. Wound healing assay and matrigel invasion assay were utilized to assess the migration and invasion ability, respectively. 7. The data were analyzed using Prism 5.0 software and expressed as mean ± SD. Statistical significance was assessed using independent two-tailed Student t-tests or Mann-Whitney’s test for independent data. Disease free survival and overall survival were analyzed using Kaplan-Meier survival curves with Gehan-BreslowWilcoxon test for significance. Difference of P < 0.05 was considered significant for the statistical tests.Results 1. Comparison of the expression of CD44 in metastatic/recurrent ovarian cancer tissues with primary counterpart, and the correlation of CD44 with the survival of patients.(1) The expression level of CD44 in primary, metastasis and recurrence ovarian cancer tissue were 1.43, 2.06 and 2.04, respectively(P value of metastatic vs. primary < 0.05; P value of recurrent vs. primary < 0.05).(2) A significant tendency of CD44 overexpression towards unfavorable prognosis was displayed in analysis of both overall survival and disease free survival(P < 0.05). 2. The expression of CD44 in ovarian ancer cell lines. The western blot results demonstrated that both the drug sensitive and drug resistant cell lines presented a ubiquitous level of CD44 expression. Moreover, SKOV-3TR and OVCAR8 TR expressed significantly higher levels of CD44 than parental sensitive cell lines, SKOV-3 and OVCAR8(P < 0.05). 3. The detection of CD44 in the tumor recurrence of human ovarian cancer xenograft model during paclitaxel treatment. No significant difference was observed between low-dosage(10 mg/kg) paclitaxel treated nude mice and saline treated ones(P > 0.05); however, there is a slight increase of CD44 in 20 mg/kg paclitaxel treated mice, and dramatic overexpression of CD44 in mice dosed with 25 mg/kg paclitaxel(P < 0.05). 4. The influnence of knockdown of CD44 by sh RNA in cell growth and the spheroid formation in 3D culture of ovarian cancer cells.(1) OVCAR8, OVCAR8 Lentivirus only, OVCAR8Non-specific sh RNA and OVCAR8CD44 sh RNA were successfully established. There was no detectable protein level of CD44 in OVCAR8CD44 sh RNA.(2) There was obvious growth inhibition in OVCAR8CD44 sh RNA(P < 0.05) and no significant difference in cell growth among other control cell lines(OVCAR8, OVCAR8 Lentivirus only, and OVCAR8Non-specific sh RNA), P > 0.05.(3) After 7-day culture in 3-D environment, which mimics in vivo settings, the diameter of spheroids formed by OVCAR8CD44 sh RNA was relatively smaller than other cell spheroids(P < 0.05). 5. The impact of CD44 in the drug sensitivity in ovarian cancer cells. The results showed minimal survival of OVCAR8CD44 sh RNAin medium containing 0.006 μM paclitaxel, while a substantial number of OVCAR8, OVCAR8 Lentivirus only, and OVCAR8Non-specific sh RNAcells were still able to tolerate this level of paclitaxel exposure. 6. The influence of CD44 in the mobility and invasion activity of ovarian cancer cells.(1) During the 24-hour incubation, the relative migratory distances of SKOV-3TR cells transfected with CD44 esi RNA were significantly suppressed compard with the control groups(P < 0.05).(2) The average numbers of CD44 esi RNA(36 n M and 54 n M) treated OVCAR8 TR cells invading through the matrigel were significantly lower in contrast with the blank control and the non-specific si RNA groups(P < 0.05).Conclusions: Up-regulation of CD44 represents a crucial event in the development of metastasis, recurrence, and drug resistance to current treatments in ovarian cancer.Section II CD44 is a direct target of mi R-199a-3p and contributes to aggressive progression in osteosarcomaObjective We sought to investigate the role of CD44 in osteosarcoma. We also aim to evaluate the regulation of CD44 by micro RNA in osteosarcoma progression.Methods 1. We constructed a human osteosarcoma tissue microarray with 114 patient tumor specimens from Massachusetts General Hospital(1992-2010), including tumor tissues from primary, metastatic, and recurrent stages, and determined the expression of CD44 by immunohistochemistry. 2. We performed microarray-based mi R profiling of human osteosarcoma cells aompared with osteoblasts. 3. Expression of mi R targeted gene protein CD44 was evaluated by Western blot. 4. MTT Assay was used to evaluate drug sensitivity in vitro. 5. The statistic analysis was similar to the methods used in Section I.Results 1. The expression of CD44 in osteoblasts, osteosarcoma cell lines and osteosarcoma tissues. There was modest expression of CD44 in HOB-c and minimal expression of CD44 in NHOst osteoblast cell lines. However, KHOS and U-2OS exhibited significantly higher expression of CD44(P < 0.05). CD44 was also expressed in osteosarcoma tissues. 2. Comparison of the expression of CD44 in metastatic/recurrent osteosarcoma tissues with primary counterpart, and the correlation of CD44 with the survival of patients and chemotherapy response.(1) The expression level of CD44 in metastasis and recurrence osteosarcoma tissue were significantly higher than the primary ttissue(P < 0.05).(2) A significant tendency of CD44 overexpression towards unfavorable prognosis was displayed in analysis of overall survival(P < 0.05).(3) Poor response osteosarcoma patients demonstrated a significantly higher level of CD44 expression, in comparison with good response counterparts(P < 0.05). 3. The correlation of CD44 with mi R-199a-3p in osteosarcoma.(1) Micro RNA.org and Target Scan databases were employed to search that one of the top ranked and highest scoring mi Rs was mi R-199a-3p. Further analysis showed that the alignment of predicted mi R-199a-3p target sequences in CD44 m RNA reside at nucleotide 74 to 91 from the start of the CD44 3′-UTR.(2) The expression of mi R-199a-3p was dramatically decreased in osteosarcoma cell lines(U-2OS and KHOS) in comparison with normalosteoblast(HOB-c and NHOst) cells(P < 0.05).(3) mi R-199a-3p repressed CD44 expression in both a dose-dependent and time-dependent manner. 4. The impact of mi R-199a-3p in the drug sensitivity of osteosarcoma. Transfection of mi R-199a-3p significantly restored the sensitivity to chemotherapeutic drug doxorubicin in U-2OS compared with the control groups(P < 0.05).Conclusions: Taken together, these results suggest that the CD44/mi R-199a-3p axis plays an important role in the development of metastasis, recurrence, and drug resistance of osteosarcoma.Section Ш Facial layer-by-layer engineering of upconversion nanoparticles for gene delivery: NIR initiate FRET trackingand overcoming drug resistance in ovarian cancerObjective We report the first use of facial layer-by-layer engineered upconversion nanoparticles(UCNPs) for near infrared initiated tracking and delivery of small interfering RNA(si RNA) to enhancechemotherapy efficacy by silencing MDR gene and desensitizing resistant ovarian cancer cells to drug.Methods 1. To obtain the nanoparticle core, Na YF4:Yb, Er UCNPs were synthesized via thermal decomposition method. To enable gene delivery, the naked UCNPs were then surface functionalized with biocompatible polymers using polyacrylic acid(PAA) and cationic poly(ethylenimine)(PEI). 2. The morphology and size of the UCNP, UCNP/PAA and UCNP/PAA/PEI were accessed by transmission electron microscopic(TEM). 3. The zeta potential was measured to confirm the success in surface functionalization by after assembly of each layer. 4. Upconversion fluorescence property was studied by fluorescence spectroscopy using 980 nm laser as excitation source. 5. Gel electrophoresis assay and Pico Green assay were employed to explore the loading capacity of the delivery system. 6. Intracellular attachment and release of MDR1 si RNA were detected by near infrared initiated fluorescence resonance energy transfer occurs between donor UCNPs and acceptor fluorescence dye labeled MDR1 si RNA. 7. Western blot determined the expression of Pgp in ovarian cancer cell line. 8. Chemotherapeutic efficacy in vitro was determined by MTT assay. 9. The statistic analysis was similar to the methods used in Section I.Results 1. Preparation of upconversion nanoparticle(UCNP) based gene delivery system: UCNP, UCNP/PAA and UCNP/PAA/PEI.(1) The zeta-potential of UCNP with negatively charged PAA coating(UCNP/PAA) was measured as-30 m V and jumped to +41 m V after positive PEI coating(UCNP/PAA/PEI). These discrete zeta-potential values alternate between negative for UCNP/PAA and positive for UCNP/PAA/PEI indicate the successful layer-by-layer assembly of PAA and PEI polymers with counter charge.(2) Under 980 nm near infrared laserexcitation, UCNP/PAA/PEI displayed the emission patterns with major emission peaks at around 530 nm and 650 nm. 2. The nucleacid encapsulation efficiency of UCNP/PAA/PEI(1) The nucleacid encapsulation efficiency and loading capacity of UCNP/PAA/ PEI were determined to be 97.264 % and 0.341 μg/μg.(2) The UCNP/PAA/PEI delivery vehicle can effectively deliver MDR1 si RNA,micro RNA Let-7 and GFP plasmid into the cells. 3. The detection of intracellular attachment and release of MDR1 si RNA with UCNP/PAA/PEI.(1) After 3h-incubation, the fluorescence resonance energy transfer(FRET) suggests that MDR1 si RNA were delivered into cells and have not yet been released form the UCNP/PAA/PEI.(2) The disassociation of MDR1 si RNA from delivery platform UCNP/PAA/PEI after pro-longed incubation time could be accessed by the loss of FRET. 4. The impact of UCNP/PAA/PEI/MDR1 si RNA complex to the chemotherapeutic efficacy of drug resistent ovarian cance cells.(1) UCNP/PAA/PEI nanoparticle system could successfully deliver the MDR1 si RNA into the cells, knockdown the protein level of its targeted Pgp(P < 0.05).(2) Ovarian cacner cells could tolerate UCNP/PAA/PEI and UCNP/PAA/PEI/ MDR1 si RNA complex well during transfection.(3) The paclitaxel IC50 values of OVCAR8 TR, OVCAR8 TR pre-treated with UCNP/PAA/PEI or UCNP/PAA/PEI/MDR1 si RNA complex were calculated to be 0.91, 0.80 and 0.59 μM. After transfection of MDR1 si RNA utilizing UCNP/PAA/ PEI, the IC50 value decreased by 1.5-fold compared to untreated OVCAR8 TR cells(P < 0.05).Conclusions: In this proof-of-concept study, layer-by-layer engineered UCNP/PAA/PEI based delivery system was successfully developed.