Figure 1.
ALDH1A1 status correlates with platinum resistance in ovarian cancer cells.
Isogenic cell lines (A2780- platinum sensitive & A2780/CP70- platinum resistant) were evaluated for platinum response using drug sensitivity assay as described in M&M section (A). To assess the percent of ALDH+ cells, in A2780 and A2780/CP70 cells ALDEFLUOR assay was performed by using BODIPY-aminoacetaldehyde (BAAA) as substrate for ALDH enzyme, after 40 minutes incubation at 37°C flow cytometry analysis was performed (B) and western blot data shows representing levels of ALDH1A1 isozyme in these cells (C).
Figure 2.
ALDH1A1 status correlates with progression free survival in ovarian cancer patients.
Ascites from 15 consecutive patients with primary advanced stage III/IV ovarian cancer and 2 from benign (Miegs syndrome) was obtained and analyzed for percentage of ALDH+ cells through ALDEFLUOR assay. The histogram in inset shows the percent of ALDH+ cells in benign and malignant ascites (A). The clinicopathologic information from the ovarian cancer patients were correlated with the percent of ALDH+ cells in their ascites and evaluated progression free survival with ALDHHIGH (>15% ALDH) to ALDHLOW (<15% ALDH) patients (3 vs. 9 months, respectively; p<0.01) (B).
Figure 3.
ALDH+ phenotypes demonstrate cancer stem cell properties.
A2780/CP70 cells were sorted into ALDH− and ALDH+ phenotypes and evaluated for their abilities for invasion using Matrigel invasion chambers (A), quantitative data as represented (B) and colony formation potential in the presence and absence of carboplatin (20 µM) were assessed using soft agar colony formation assays (C). In order to determine possible mechanism for cancer stem cell characteristics in ALDH+ cells, we evaluated multiple markers of “stemness”. A2780/CP70 cells were sorted into ALDH− and ALDH+ phenotypes, total RNA was isolated, cDNA was prepared and real-time quantitative RT-PCR was performed (D). **indicates statistical significance (p<0.01).
Figure 4.
ALDH1A1 down regulation differentially affects on cancer stem cell properties.
Lentiviral vectors expressing ALDH1A1 specific shRNAs or nontargeting shRNAs were transfected into A2780/CP70 cells and its effect on stem-like cell properties were evaluated for invasive potential using Matrigel invasion chambers (A), quantitative data as represented (B), clonogenic potential using soft agar colony formation (C, and carboplatin dependent growth inhibition by CellTiter-Glo Luminescent Cell Viability Assay (D). Statistical significance was evaluated using student’s t-test.
Figure 5.
ALDH1A1 downregulation leads to lower KLF4 & p21 expression with altered cell-cycle profile.
ALDH1A1 proficient and deficient A2780/CP70 cells were evaluated for expression of stem-like cell marker KLF4 and cell cycle checkpoint protein p21 by RT-PCR and Western blot analysis (A). Cell cycle distributions of cells were analyzed after fixing the cells in 70% methanol and propidium iodide staining followed by flow cytometry (B). Apoptotic cells were detected after staining the cells with APC-Annexin V and 7-AAD according to the manufacturer’s instructions and analyzed by flow cytometry (C).
Table 1.
The human Cancer Drug Resistance & Cell Cycle RT2 Profiler PCR Array results were compared between ALDH1A1 knockdown and controls.
Figure 6.
ALDH1A1 status alters cell cycle checkpoints and DNA repair networks.
ALDH1A1 proficient (control) and deficient (shALDH1A1) A2780/CP70 cells were assessed for their abilities to repair carboplatin induced single strand breaks by looking at time dependent induction of PARP-1 protein levels by western blots (A), densitometry of blots by Image J (B) and total PARP activity was assayed by measuring PAR levels (C). To assess the ALDH1A1 dependent expression DDR and repair proteins, whole cell lysates were normalized for total proteins and western blot analysis were performed using antibodies as represented (D).