J Korean Med Sci.  2016 Mar;31(3):360-370. 10.3346/jkms.2016.31.3.360.

Dual-Blocking of PI3K and mTOR Improves Chemotherapeutic Effects on SW620 Human Colorectal Cancer Stem Cells by Inducing Differentiation

• Kim MJ
• Koo JE
• Han GY
• Kim B
• Lee YS
• Ahn C
• Kim CW

• KMID: 2363495
• DOI: http://doi.org/10.3346/jkms.2016.31.3.360

Abstract

Cancer stem cells (CSCs) have tumor initiation, self-renewal, metastasis and chemo-resistance properties in various tumors including colorectal cancer. Targeting of CSCs may be essential to prevent relapse of tumors after chemotherapy. Phosphatidylinositol-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) signals are central regulators of cell growth, proliferation, differentiation, and apoptosis. These pathways are related to colorectal tumorigenesis. This study focused on PI3K and mTOR pathways by inhibition which initiate differentiation of SW620 derived CSCs and investigated its effect on tumor progression. By using rapamycin, LY294002, and NVP-BEZ235, respectively, PI3K and mTOR signals were blocked independently or dually in colorectal CSCs. Colorectal CSCs gained their differentiation property and lost their stemness properties most significantly in dual-blocked CSCs. After treated with anti-cancer drug (paclitaxel) on the differentiated CSCs cell viability, self-renewal ability and differentiation status were analyzed. As a result dual-blocking group has most enhanced sensitivity for anti-cancer drug. Xenograft tumorigenesis assay by using immunodeficiency mice also shows that dual-inhibited group more effectively increased drug sensitivity and suppressed tumor growth compared to single-inhibited groups. Therefore it could have potent anti-cancer effects that dual-blocking of PI3K and mTOR induces differentiation and improves chemotherapeutic effects on SW620 human colorectal CSCs.

Keyword

Cancer Stem Cells; PI3K; mTOR; Drug Resistance; Differentiation Therapy

MeSH Terms

AC133 Antigen/genetics/metabolism
Animals
Antineoplastic Agents/pharmacology/therapeutic use
Cell Differentiation/*drug effects
Cell Line, Tumor
Cell Survival/drug effects
Chromones/pharmacology/therapeutic use
Colorectal Neoplasms/drug therapy/metabolism/pathology
Humans
Imidazoles/pharmacology/therapeutic use
Male
Mice
Mice, Inbred BALB C
Mice, Nude
Morpholines/pharmacology/therapeutic use
Neoplastic Stem Cells/cytology/drug effects/metabolism
Paclitaxel/pharmacology/therapeutic use
Phosphatidylinositol 3-Kinases/*antagonists & inhibitors/metabolism
Quinolines/pharmacology/therapeutic use
SOXB1 Transcription Factors/genetics/metabolism
Signal Transduction/*drug effects
Sirolimus/pharmacology/therapeutic use
TOR Serine-Threonine Kinases/*antagonists & inhibitors/metabolism
Xenograft Model Antitumor Assays
AC133 Antigen
Antineoplastic Agents
Chromones
Imidazoles
Morpholines
Paclitaxel
Phosphatidylinositol 3-Kinases
Quinolines
SOXB1 Transcription Factors
Sirolimus
TOR Serine-Threonine Kinases

Figure

• 

  Fig. 1 Stemness properties of sorted SW620 CD133+ cells. (A) The mRNA expression of stemness and differentiation marker in SW620 CD133+ and CD133–cells were measured by RT-PCR. β-Actin was used as a loading control. For comparisons, the relative value for markers of CD133–cells was considered to be “1”. (B) Self-renewal ability of sorted cells was analyzed by sphere formation assay. Pictures were taken at ×40 magnification. Scale bar = 100 µm. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed (*P < 0.001).

• 

  Fig. 2 Stemness and differentation properties of PI3K and/or mTOR inhibitors treated SW620 CD133+ cells. (A) The mRNA expression of stemness and differentiation markers after inhibition of PI3K and/or mTOR was measured by RT-PCR. β-Actin was used as a loading control. For comparisons, relative intensity for markers of DMSO-treated CD133+ cells was defined as “1”. *P < 0.001. (B) The protein expression of CD133 was measured by Immunofluorescence assay (IF). CD133 was stained in red. (C) The protein expression of CEA was measured by IF. CEA were stained in green, respectively. In each experiment (B and C), the nuclei were counterstained with DAPI (blue). For comparisons, relative intensity of Corrected Total Cell Fluorescence (CTCF) for CD133 and CEA of DMSO-treated CD133+ cells was defined as “1”. Data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed (*P < 0.001, “N/S” means “statistically not significant”). Pictures were taken at ×400 magnification. Scale bar = 10 μm.

• 

  Fig. 3 The drug sensitivity of inhibited SW620 CD133+ cells. (A) Inhibited SW620 CD133+ cells were treated with paclitaxel (100 nM) for 24 hours and cell viability was assessed by CCK-8 assay. For comparisons cell viability of DMSO treated CD133+ cells was 100%. (B) Self-renewal ability of inhibited SW620 CD133+ cells treated with paclitaxel and self-renewal ability was assessed by sphere formation assay. (C) Differentiation marker (CEA) expression of inhibited CSCs treated with paclitaxel. The protein expression of CEA was assessed by IF. For the comparison, relative intensity of CTCF for CEA of DMSO treated CD133+ cells was defined as “1”. All data are expressed as the mean ± standard error of the mean (SEM) of three independent experiments performed (*P < 0.05, †P < 0.01, ‡P < 0.001, “N/S” means “statistically not significant”). Pictures were taken at ×400 magnification. Scale bar = 10 μm.

• 

  Fig. 4 Xenograft tumorigenecity assay of inhibitors treated SW620 CD133+ cells before and after injection of anti-cancer drug. (A) Comparison of tumorigenecity of inhibitor treated SW620 CD133+ cells in BALB/c nude mice. Balb/c nude mice were subcutaneously injected with sorted SW620 CD133+ cells. The left flank was injected with untreated CD133+ cells, while the right flank was injected with CD133+ cells treated with each inhibitor. Anti-cancer drug (paclitaxel) was injected since 28 days. (B) Tumor volume (mm3) of xenografts from Balb/c mice. The change in tumor volume was checked for each group. DMSO treated group was used as a control. Data are expressed as the mean ± standard error of the mean (SEM) of mice for each group.

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