Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: Hypoxia and chemotherapy induce HO-1 expression in different PC cell lines. A comparative analysis of HMOX-1 expression between normal prostate tissues group and tumor prostate tissues group using the dataset with accession number GSE30994, and between hormone resistant tissues group and the normal prostate tissues group using the dataset with the accession number GSE200879 from the GEO database (A) . Western blot and densitometry analysis showed that chemotherapy (Doc) induced HO-1 expression in DU145 (10 nM), LNCaP (10 nM), and C4–2B (0.5 μM) after 24 h treatment (B) . 24 h hypoxia (Hyp) induced HO-1 expression in LNCaP and DU145 cells (C) . qRT-PCR analysis showed that HMOX-1 gene expression was induced after 6 h and 18 h hypoxia but only significant at the 18 h exposure in DU145 and LNCaP cells (D, E) . qRT-PCR showed that 24 h treatment with Doc (10 nM) significantly induced HMOX-1 gene expression compared to non-treated control (F) . Hemin (10 μM) was used to validate the inducibility of HO-1 in our PC cell lines represented by DU145 (G) . Confocal analysis further confirmed our finding that 24 h treatment with Doc (20nM) induced HO-1 levels in LNCaP and DU145 cells and that using NAC (1 mM), an ROS scavenger, for 24 h treatment significantly reduced HO-1 levels in DU145 cells (H, I) . (n=3, **** = P<0.0001, *** = P<0.0001, ** = P<0.001, * = P<0.05).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Expressing, Western Blot, Quantitative RT-PCR, Gene Expression, Control

Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: HO-1 inhibition decreases cellular viability and increases sensitivity to Doc in different PC cells. MTT analysis after 48 h treatment showed significant increase in PC cells sensitivity to Doc (0.5 μM) and significant decrease in cellular viability when using ZnPP (5 μM) and SnPP (15 μM) as our HO-1 inhibitors in LNCaP cells (A) , DU145 (B) , and C4–2B (C) . HO-1 inhibition had the same effect under 48 h hypoxic condition compared to normoxic conditions measured by MTT assay in LNCaP with 10 nM Doc and 5 μM ZnPP (D) , DU145 with 10 nM Doc and 5 μM ZnPP (E) , and C4–2B cells with 0.5 μM Doc and 5 μM ZnPP (F) . HO-1 KD, confirmed by western blot analysis employing CoPP (10 μM) as the HO-1 inducer, resulted in a significant decrease in cellular viability when seeded at different densities, 3000, 5000, and 10,000 cells, in comparison to the parental cells at the same seeding density measured by the MTT assay after 48 h in LNCaP cells (G) and in DU145 cells (H) . HO-1 KD increased PC cells sensitivity to Doc (10 nM) in LNCaP cells after 48 h treatment using MTT assay and normalized to own control (I) . DU145 HO-1 KD cells showed the same increase in sensitivity to Doc (10 nM) when compared to parental response and normalized to parental control after 48 h treatment (J) . LNCaP HO-1 Tet-On, confirmed by western blot analysis after 48 h treatment with Dox (0.5 μM and 0.75 μM) and compared to parental cells treated with CoPP (10 μM), further confirmed our findings and HO-1 induction resulted in an increase in PC cells resistance to the Doc (10 nM) treatment measured by the MTT assay after 48 h treatment represented in the bar graph (K) . Combination index (CI) values between Doc alone and Doc combined with ZnPP or SnPP are depicted showing synergistic effect (CI<1) or additive effect (CI=1). (n=3, **** = P<0.0001, *** = P<0.0001, ** = P<0.001, * = P<0.05).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Inhibition, MTT Assay, Western Blot, Comparison, Control

Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: Combinational therapy of HO-1 and Doc enhances the response to apoptosis induced by Doc. Flow cytometry analysis revealed that the combined therapy of Doc (10 nM) and ZnPP (5 µM) significantly increased the number of apoptotic cells compared to each treatment separately and to the untreated control cells after 24 h treatment in DU145 (A) and LNCaP cells (B) . (n=3, **** = P<0.0001, ** = P<0.001).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Flow Cytometry, Control

Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: HO-1 inhibition increases ROS production in PC cells and disrupts the glutathione cycle. DCFDA assay demonstrated a significant increase in ROS levels with each treatment separately, ZnPP (5 µM) and Doc (0.5 µM) and was further induced with combinational treatment resulting in a significant increase in ROS levels compared to each treatment alone and to the control in LNCaP cells (A) and in C4-2B cells (B) following a 24 h treatment. For ROS quantification, ImageJ software was used. Employing NAC (1 mM), an ROS scavenger, significantly increased cell survival and reduced the efficacy of Doc (5 nM) treatment and ZnPP (2 μM) in LNCaP cells measured by MTT proliferation assay after 48 h treatment (C) . Combining HO-1 inhibitor, ZnPP (5 µM), with Doc (10 nM) resulted in disruption of glutathione homeostasis in DU145 cells following a 48 h treatment (D) . (n=3, **** = P<0.0001, *** = P<0.0001, * = P<0.05).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Inhibition, Control, Software, Proliferation Assay, Disruption

Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: HO-1 inhibition disrupts the STAT1 signaling pathway. qRT-PCR analysis in LNCaP cells confirmed the higher expression level of STAT1 gene with the 24 h Doc (10 nM) treatment compared to untreated control cells (A) . MTT assay following a 48 h treatment showed that using Fluda (15 µM), a STAT1 specific inhibitor, significantly decreased PC cell survival when combined with either Doc (10 nM) or ZnPP (5 µM) in LNCaP cells under normoxia (B) , and in DU145 cells under both normoxic and hypoxic conditions (C, D) . qRT-PCR analysis revealed a significant decrease in HMOX-1 gene expression levels after 24 h treatment with Fluda (25 µM), alone or in combination with Doc (10 nM), in DU145 cells (E) . (n=3, **** = P<0.0001, *** = P<0.0001, * = P<0.05).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Inhibition, Quantitative RT-PCR, Expressing, Control, MTT Assay, Gene Expression

Journal: Frontiers in Oncology

Article Title: Targeting heme degradation pathway augments prostate cancer cell sensitivity to docetaxel-induced apoptosis and attenuates migration

doi: 10.3389/fonc.2024.1431362

Figure Lengend Snippet: HO-1 HO-1 inhibition reduces PC cell migratory potential and affects the EMT capability in PC cells. Scratch assay showed that HO-1 inhibition and HO-1 KD cells significantly reduced PC cells migration. Treating DU145 cells with ZnPP (3 µM) and imaging the scratch at 0, 24, and 48 h post treatment significantly reduced PC cells migration, which correlates with percent gap closure, compared to the control untreated cells (A, B) . LNCaP cells treated with ZnPP (3 µM) and LNCaP HO-1 KD cells, both imaged at 0, 24, and 48 h, exhibited similar reduction in the rate of gap closure compared to non-treated control cells (C, D) . Representative Images of the control and treated groups at different time points, 0, 24, and 48 h, of the scratch assay (A, C) . The rate of wound closure at different time points (B, D) . Western blot and densitometry analysis showed decreased levels of EMT markers, Vimentin and Snail, with HO-1 inhibition in DU145 cells after 24 h treatment with Doc (10 nM) and ZnPP (5 µM) (E, F) . (n=3, **** = P<0.0001, * = P<0.05).

Article Snippet: DU145 cells (RRID: CVCL_0105) and LNCaP cells (RRID: CVCL_0395) were transduced with lentivirus obtained from VectorBuilder Inc., Chicago, IL, USA, at a multiplicity of infection (MOI) ranging from 5 to 10.

Techniques: Inhibition, Wound Healing Assay, Migration, Imaging, Control, Western Blot

Journal: Cancer Biomarkers

Article Title: Transcription factor KLF13 inhibits AKT activation and suppresses the growth of prostate carcinoma cells

doi: 10.3233/CBM-181196

Figure Lengend Snippet: Overexpression of KLF13 inhibits cell proliferation in vitro . (A and B) PC3 and DU145 cells transfected with pTRIPZ-KLF13 or pTRIPZ-vector were cultured with (Tet+) or without (Tet-) doxycycline (0.25 mg/mL) for 48 h. KLF13 expression was verified by Western blot. The viability of cells was assessed by CCK8 assay (OD value 450 nm) at the indicated time. (C and D) Colony formation rates of PC3 and DU145 cells treated with or without doxycycline for 10 days. Each value represents the mean ± SD of three independent experiments. P * ⁣ * ⁣ * < 0.001.

Article Snippet: Human prostate cancer cell lines, DU145 and PC3, were obtained from Renji Hospital Clinical Stem Cell Research Center (Shanghai, China).

Techniques: Over Expression, In Vitro, Transfection, Plasmid Preparation, Cell Culture, Expressing, Western Blot, CCK-8 Assay