Quercetin abrogates chemoresistance in melanoma cells by modulating ΔNp73

<p>Abstract</p> <p>Background</p> <p>The alkylating agent Dacarbazine (DTIC) has been used in the treatment of melanoma for decades, but when used as a monotherapy for cancer only moderate response rates are achieved. Recently, the clinical use of Temozolomide (TMZ) has become the more commonly used analog of DTIC-related oral agents because of its greater bioavailability and ability to cross the blood brain barrier. The response rates achieved by TMZ are also unsatisfactory, so there is great interest in identifying compounds that could be used in combination therapy. We have previously demonstrated that the bioflavonoid quercetin (Qct) promoted a p53-mediated response and sensitized melanoma to DTIC. Here we demonstrate that Qct also sensitizes cells to TMZ and propose a mechanism that involves the modulation of a truncated p53 family member, ΔNp73.</p> <p>Methods</p> <p>DB-1 melanoma (p53 wildtype), and SK Mel 28 (p53 mutant) cell lines were treated with TMZ (400 μM) for 48 hrs followed by Qct (75 μM) for 24 hrs. Cell death was determined by Annexin V-FITC staining and immunocytochemical analysis was carried out to determine protein translocation.</p> <p>Results</p> <p>After treatment with TMZ, DB-1 cells demonstrated increased phosphorylation of Ataxia telangiectasia mutated (ATM) and p53. However, the cells were resistant to TMZ-induced apoptosis and the resistance was associated with an increase in nuclear localization of ΔNp73. Qct treatment in combination with TMZ abolished drug insensitivity and caused a more than additive induction of apoptosis compared to either treatment alone. Treatment with Qct, caused redistribution of ΔNp73 into the cytoplasm and nucleus, which has been associated with increased p53 transcriptional activity. Knockdown of ΔNp73 restored PARP cleavage in the TMZ treated cells, confirming its anti-apoptotic role. The response to treatment was predominantly p53 mediated as the p53 mutant SK Mel 28 cells showed no significant enhancement of apoptosis.</p> <p>Conclusion</p> <p>This study demonstrates that Qct can sensitize cells to TMZ and that the mechanisms of sensitization involve modulation of p53 family members.</p

Novel 3D co-culture model for epithelial-stromal cells interaction in prostate cancer.

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction

Novel 3D co-culture model for epithelial-stromal cells interaction in prostate cancer.

Paracrine function is a major mechanism of cell-cell communication within tissue microenvironment in normal development and disease. In vitro cell culture models simulating tissue or tumor microenvironment are necessary tools to delineate epithelial-stromal interactions including paracrine function, yet an ideal three-dimensional (3D) tumor model specifically studying paracrine function is currently lacking. In order to fill this void we developed a novel 3D co-culture model in double-layered alginate hydrogel microspheres, incorporating prostate cancer epithelial and stromal cells in separate compartments of the microspheres. The cells remained confined and viable within their respective spheres for over 30 days. As a proof of principle regarding paracrine function of the model, we measured shedded component of E-cadherin (sE-cad) in the conditioned media, a major membrane bound cell adhesive molecule that is highly dysregulated in cancers including prostate cancer. In addition to demonstrating that sE-cad can be reliably quantified in the conditioned media, the time course experiments also demonstrated that the amount of sE-cad is influenced by epithelial-stromal interaction. In conclusion, the study establishes a novel 3D in vitro co-culture model that can be used to study cell-cell paracrine interaction

Co-culture of prostate cancer epithelial cells and normal prostate stromal cells decreased sE-cad secretion by the epithelial cells.

<p>sE-cad level was measured by ELISA following co-culture for 30 days. SB, microcapsules with stromal cells at inner core and blank outer layer. BV, microcapsules with blank inner core and C4-2 vector cells in outer layer. BP, microcapsules with blank inner core and C4-2 PKD1 cells in outer layer. SV, microcapsules with stromal cells at inner core and C4-2 vector cells in outer layer. SP, microcapsules with stromal cells at inner core and C4-2 PKD1 transfected cells in outer layer. P values were calculated by Student’s t-test. Each column represents the average of four parallel experiments. Error bar represents the standard error.</p

Whereas normal stromal cells do not secret sE-Cad in microcapsules, prostate cancer epithelial cells do.

<p>A, cells were cultured in 3D environment for 6 days. BS, microcapsules with blank inner core and stromal cells in outer layer. BV, microcapsules with blank inner core and C4-2 vector cells in outer layer. BP, microcapsules with blank inner core and C4-2 PKD1 cells in outer layer. sE-Cad level was measured by ELISA. B, cells were cultured in 2D environment (petri dish). sE-Cad level was measured by ELISA and normalized to reflect the secretion of 10⁷ cells for each cell line P values were calculated by Student’s t-test. Each column represents the average of three (2D culture) or four (3D culture) parallel experiments. Error bar represents the standard error.</p

Different lines of prostate cells remained viable in microcapsules for over a month.

<p>BS, microcapsules with blank inner core and stromal cells at outer layer. PS, microcapsules with C4-2 PKD1 cells at inner core and stromal cells at outer layer. Green, live cells at inner core. Red, live cells at outer layer. Blue, dead cells. Scale bar, 100µm.</p

sE-Cad secretions in different human cell types.

<p>sE-Cad level was measured by ELISA and was normalized to reflect the secretion of 10⁷ cells for each cell line. sE-Cad level of metastatic cell lines (MCF-7, LnCap and PC3) were compared to HEK293T (as a representative of normal cell lines) and P values were calculated by Student’s t-test. Each column represents the average of three parallel experiments. Error bar represents the standard error.</p

Effects of Allogeneic Bone Marrow Derived Mesenchymal Stromal Cell Therapy on Voiding Function in a Rat Model of Parkinson Disease

Purpose: Cellular therapy induced transient urodynamic improvement in a rat model of Parkinson disease in which bladder dysfunction was noted after unilateral injection of 6-hydroxydopamine into the medial forebrain bundle. We sought to prolong the effect by injecting allogeneic rat bone marrow mesenchymal stromal cells before and after microencapsulation into the substantia nigra pars compacta.Materials and Methods: Female rats underwent unilateral stereotactic injection of 6-hydroxydopamine in the medial forebrain bundle. Injection was performed in the ipsilateral substantia nigra pars compacta using vehicle alone or vehicle with nonmicroencapsulated or microencapsulated rat bone marrow derived mesenchymal stromal cells. Rats were evaluated by cystometry 7, 14, 28 and 42 days after treatment. Brains were extracted for immunostaining.Results: At 42 days the nonmicroencapsulated group had lower threshold and intermicturition pressure, spontaneous activity and AUC than vehicle treated animals. Rats that received microencapsulated cells had lower threshold pressure at 28 days and lower spontaneous activity at 42 days than vehicle treated rats. Microencapsulated and nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells were noted in the substantia nigra pars compacta up to 42 days after transplantation. At 42 days tyrosine hydroxylase positive neurons were more numerous in the substantia nigra pars compacta of the nonmicroencapsulated group, followed by the microencapsulated and vehicle treated groups.Conclusions: Urodynamic effects of the 6-hydroxydopamine lesion persisted up to 42 days after vehicle injection. Transplantation of nonmicroencapsulated rat bone marrow derived mesenchymal stromal cells improved urodynamic pressure by 42 days after treatment more markedly than microencapsulated cells. This was associated with more tyrosine hydroxylase positive neurons in the treated substantia nigra pars compacta of the nonmicroencapsulated group, suggesting that functional improvement requires a juxtacrine effect.American Urological Association FoundationAmerican Urological Association Southeastern Section Research Scholar Endowment FundWake Forest Univ, Bowman Gray Sch Med, Wake Forest Inst Regenerat Med, Winston Salem, NC USANYU, Dept Urol, New York, NY USAUniversidade Federal de São Paulo, Div Urol, São Paulo, BrazilFukushima Med Univ, Dept Urol, Sch Med, Fukushima, JapanUniversidade Federal de São Paulo, Div Urol, São Paulo, BrazilWeb of Scienc