Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Schematic representation of protoporphyrin IX (PpIX) generation in normal and cancer cells. Upon exogenous stimulation with 5-ALA, cells generate PpIX via the haem biosynthesis pathway. PpIX is subsequently converted to haem by FECH or transported outside the cells through efflux receptors such as ABCB1. As oncogenic transformation activates enzymes of the haem biosynthesis pathway, cancer cells generate PpIX more efficiently than normal cells. As the Ras/MEK pathway promotes PpIX conversion to haem and PpIX efflux through ABCB1, MEK inhibition further enhances PpIX accumulation in cancer cells.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Transformation Assay, Inhibition

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Oncogenic Ras/MEK regulates PpIX accumulation via ABCB1 and FECH. ( A ) Representative overlay histograms showing surface ABCB1 expression in NIH3T3 cells and RasV12 cells treated with or without different concentrations of MEK inhibitor, U0126. ( B ) Plot shows mean ± SD mean fluorescence intensity (MFI) from 3 independent experiments. ( C ) Mean ± SD relative FECH activity in NIH3T3 cells and RasV12 cells treated with or without different concentrations of U0126 from 3 independent experiments. ( D ) Representative western blot showing FECH expression and ERK phosphorylation levels in NIH3T3 and RasV12 cells treated with or without different concentrations of U0126. The relative band densities (RD) are indicated. *p < 0.01 by one-way ANOVA with Turkey's posthoc test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Expressing, Fluorescence, Activity Assay, Western Blot

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Oncogenic Ras regulates ABCB1 expression via RSKs. ( A ) Representative western blot showing RSK phosphorylation in RasV12 cells with or without MEK inhibition. The relative band densities (RD) are indicated. ( B ) (Top) Representative gel image showing RSK1, RSK2, and RSK3 cDNA levels in RasV12 cells treated with or without various RSK siRNAs. (Bottom) Representative western blot showing RSK2 expression in RasV12 cells treated with or without various RSK siRNAs. The relative band densities (RD) are indicated. Mean ± SD PpIX fluorescence in RasV12 cells ( C ) transfected with siRNA against RSKs and ( D ) treated with different concentrations of SL0101, a pan-RSK inhibitor. *p < 0.01 by one-way ANOVA with Turkey's posthoc test. ( E ) Representative western blots showing ABCB1 expression in RAS V12 cells treated with different concentrations of SL0101. p-s6 expression was used to confirm RSK inhibition, and GAPDH was used as the loading control. The relative band densities (RD) are indicated.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Expressing, Western Blot, Inhibition, Fluorescence, Transfection

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Inhibiting RSKs, ABCB1, and HIF-1α enhanced PpIX accumulation in human cancer cell lines. Human cancer cell lines DLD-1, SNB-75, Hs 578 T, and MDA MB 231 were pre-treated with or without SL0101 (RSK inhibitor), Zosuquidar (ABCB1 inhibitor), or HIF 1α inhibitor for 20 h and then with 5-ALA for 4 h. Mean ± SEM PpIX fluorescence in cell lysate compared to controls is shown. *p < 0.01 by Student's t -test.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Fluorescence

Journal: Scientific Reports

Article Title: MEK reduces cancer-specific PpIX accumulation through the RSK-ABCB1 and HIF-1α-FECH axes

doi: 10.1038/s41598-020-79144-x

Figure Lengend Snippet: Reduction in PpIX accumulation through the Ras/MEK-HIF-1α-FECH and Ras/MEK-RSK-ABCB1 axes in cancer cells. Ras/MEK activation increases HIF-1α expression, which in turn increases the activity of FECH, the enzyme that catalyses the conversion of PpIX to haem. Ras/MEK activation also upregulates ABCB1 expression through RSKs, which promotes the rate of PpIX efflux.

Article Snippet: Anti-phospho-ERK-1/2 and anti-phospho RSK antibodies were purchased from Cell Signaling (Danvers, MA), anti-ABCB1 antibody from Alomone Labs (Israel), anti-FECH, anti-phospho-S6, anti-RSK2, anti-total ERK antibodies, and the FITC-tagged Anti-ABCB1 antibody from Santa Cruz Biotechnology; anti-HIF-1α antibody, and anti-GAPDH antibody were purchased from Abcam (US).

Techniques: Activation Assay, Expressing, Activity Assay