Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: CD36 is overexpressed in human CRC. (A,B) Immunoreactivity score of CD36 expression was analyzed in matched normal colon mucosa and tumor tissues from patients diagnosed with Stage I–IV CRC (TMA: n = 56, * p < 0.001 vs. normal tissue). (C) CD36 staining in matched normal colon mucosa, primary CRC, and CRC metastasis to liver and lung [representative images are shown; liver ( n = 12) and lung metastasis ( n = 5)]. (D) Correlations between FASN and CD36 was determined based on RNASeq data of CRC patient samples ( n = 22 of normal tissues and n = 215 of tumors) from The Cancer Genome Atlas. (E) Expression of FASN and CD36 in human normal colon mucosa and tumor tissues. N, normal mucosa; T, primary tumor; L, normal liver tissue; M, liver metastasis.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Expression of CD36 is selectively regulated by the level of de novo fatty acid synthesis in CRC. (A) shRNA-mediated knockdown of FASN leads to upregulation of CD36 mRNA expression in HCT116 cells (* p < 0.05). (B) TVB-3664 treatment of CRC tissue slices (18 h) selectively upregulates CD36 mRNA expression (* p < 0.05). (C) TVB-3664 treatment of Pt 93 and Pt 130 primary CRC cells increases CD36 mRNA and protein expression. (D) shRNA mediated knockdown of FASN increases CD36 protein expression in HCT116 and HT29 cells. (E) Relative mRNA expression of FASN and CD36 in intestinal tumors collected from APC/Cre and FASN +/− /APC/Cre mice. (F) FASN and CD36 protein expression in intestinal mucosa collected from Apc/Cre and Apc/Cre mice with hetero- and homo-zygous deletion of FASN. (G,H) Inhibition of FASN increases membrane-associated expression of CD36. (G) Confocal images of FASN and CD36 in control and 0.2 μM TVB-3664 treated (6 days) Pt 93 primary CRC cells. (H) Flow cytometry analysis of Pt 93 and Pt 130 primary CRC cells treated with 0.2 μM TVB-3664 (6 days) in normal and serum free media conditions. Mean fluorescence for CD36 is shown for representative data from three different experiments (** p < 0.01, * p < 0.05). (I) FA uptake in HCT116, NTC, and FASN shRNA. Cells were pre-treated with anti-CD36 antibody or vehicle for 24 h and then treated with BODIPY FL for 10 min.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, shRNA, Knockdown, Inhibition, Membrane, Control, Flow Cytometry, Fluorescence

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 is associated with decreased cellular proliferation. (A) Primary Pt 93 and Pt 130 CRC cells treated with 100 μM SSO for 6 days. Cellular proliferation assays were performed via cell count. Representative data from three experiments is shown (* p < 0.05). (B) Confocal images of FASN and CD36 in Pt 93 cells in normal and serum free media (6 days). (C) Expression of proteins associated with apoptosis and survival in HCT116 transfected with CD36 shRNAs and analyzed via western blot. (D) Cellular proliferation assay with HCT116, NTC, and CD36 shRNA (* p < 0.05 for normal medium, # p < for SFM). (E) qRT-PCR confirmation of CD36 knockdown using CD36 shRNA #2 (73%) and CD36 shRNA #4 (67%).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Cell Counting, Expressing, Transfection, Western Blot, Proliferation Assay, shRNA, Quantitative RT-PCR, Knockdown

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: SSO treatment and CD36 knockdown inhibit tumor growth in vivo . (A) Tumor volume, tumor weight, and mouse weight of control and SSO treated (20 mg/kg) mice are shown. SSO was dissolved in 10% PEG and administered in 200 μl dosages via oral gavage daily. 1.0 × 10 6 cells were injected into NU/NU mice. Treatment was initiated when tumors reached ~100 mm 3 (day 0). (B) RT-PCR analysis of HCT116 tumors showing the effect of SSO treatment on CD36, FASN, and survivin mRNA expression. (C) Tumor volume of HCT116 NTC and CD36 shRNA #2 and #4 xenografts is shown. 1.0 × 10 6 cells were injected into NU/NU mice and tumor growth was measured every 3 days. (D) H&E and Ki67 staining of HCT116 NTC and CD36 shRNA tumors. (E) Tumor volume and tumor weight of HT29 LuM3 NTC and CD36 shRNA #4 xenografts are shown. (F) mRNA expression of survivin in HT29 LuM3 xenografts (analysis of tumors from 2 mice per group is show).

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Knockdown, In Vivo, Control, Injection, Reverse Transcription Polymerase Chain Reaction, Expressing, shRNA, Staining

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: High expression of CD36 is associated with an increase in pAkt and survivin in CRC. (A) Diagram of Pt 2402 propagation after flow cytometry sorting for CD36 high and CD36 low cells. (B) Numbers of CD36 high and CD36 low Pt 2402 cells for first and second flow cytometry sorts. (C) Protein expression levels of FASN, CD36, and survivin in CD36 high and CD36 low Pt 2402 primary cells from first flow cytometry sort. (D) IHC staining for Ki67 in Pt 2402 CD36 high and CD36 low tumors. (E) Protein expression levels of FASN, CD36, pAkt, cleaved PARP, and survivin in HCT116 CRC cells, control, and CD36 overexpression.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Expressing, Flow Cytometry, Immunohistochemistry, Control, Over Expression

Journal: Frontiers in Oncology

Article Title: Inhibition of Fatty Acid Synthase Upregulates Expression of CD36 to Sustain Proliferation of Colorectal Cancer Cells

doi: 10.3389/fonc.2020.01185

Figure Lengend Snippet: Inhibition of CD36 and FASN have a synergetic effect in reducing cell proliferation. (A) Pt 93, Pt 130, and HCT116 cells were treated with SSO and TVB-3664 alone or in combination for 6 days and cell number was counted. Representative data from three experiments is shown (* p < 0.05). (B) Western blot analysis of cells treated with TVB-3664, SSO, or TVB-3664 and SSO in combination.

Article Snippet: QRT-PCR was carried out using a TaqMan Gene Expression Master Mix (#4369016) according to manufacture protocol and TaqMan probes for human CD36 (ID Hs00354519 m1), human FASN (ID Hs01005622 m1), human FATP3 (ID Hs00354519 m1), human FATP4 (Hs00192700 m1), and human GAPDH (#4333764F; Applied Biosystems).

Techniques: Inhibition, Western Blot