Abstract
BackgroundCancer stem cells (CSCs) are proposed to be responsible for high recurrence rate in cervical carcinoma. Reagents that can suppress the proliferation and differentiation of CSCs would provide new opportunities to fight against tumor recurrence. Doxycycline has been reported as a potential anti-cancer compound. However, few studies investigated its inhibitory effect against cervical cancer stem cells.MethodsHeLa cells were cultured in cancer stem cell conditional media in a poly-hema-treated dish. In this non-adhesive culture system, HeLa cells were treated with cisplatin until some cells survived and formed spheroids, which were then collected and injected into the immunodeficient mice. Cisplatin was administered every three days for five times. The tumor xenografts with CSC enrichment were cultured in cancer stem cell specific medium again to form tumorsphere, which we called HeLa-CSCs. Expression of cancer stem cell markers in HeLa-CSCs was measured by flow cytometry and qPCR. HeLa-CSCs were then treated with doxycycline. Proliferation and differentiation rates were determined by the size of spheres formed in vitro and tumor formed in vivo.ResultsWe developed a new strategy to selectively enrich CSCs from human cervical carcinoma cell line HeLa, and these HeLa-CSCs are CD133+/CD49f+ cell populations with significantly enhanced expression of stem cell markers. When these HeLa-CSCs were treated with doxycycline, the colony formation, proliferation, migration and invasion, and differentiation were all suppressed. Meanwhile, stem cell markers SOX-2, OCT-4, NANOG, NOTCH and BMI-1 decreased in doxycycline treated cells, so as the surface markers CD133 and CD49f. Furthermore, proliferation markers Ki67 and PCNA were also decreased by doxycycline treatment in the in vivo xenograft mouse model.ConclusionsCancer stem cells are enriched from sphere-forming and chemoresistant HeLa-derived tumor xenografts in immunodeficient mice. Doxycycline inhibits proliferation, invasion, and differentiation, and also induces apoptosis of these HeLa-CSCs in vitro and in vivo.
Highlights
Carcinoma of the uterine cervix is the second most common malignancy that affects women worldwide and causes high mortality; approximately 500,000 new cases and more than 270,000 deaths occur each year due to this disease [1, 2]
We developed a new strategy to selectively enrich Cancer stem cells (CSCs) from human cervical carcinoma cell line HeLa, and these HeLa-CSCs are CD133+/CD49f+ cell populations with significantly enhanced expression of stem cell markers
When these HeLa-CSCs were treated with doxycycline, the colony formation, proliferation, migration and invasion, and differentiation were all suppressed
Summary
Carcinoma of the uterine cervix is the second most common malignancy that affects women worldwide and causes high mortality; approximately 500,000 new cases and more than 270,000 deaths occur each year due to this disease [1, 2]. While it is well known that self-renewal and differentiation capacity are hallmark traits of normal stem cells (SCs), tumor cells are found to possess the high proliferative capacity and phenotypic plasticity [3] These similarities have given rise to the hypothesis of the cancer stem cells (CSCs), a subpopulation of cancer cells derived from normal SCs possessing tumorinitiating capability [6,7,8]. There were some reports suggesting CD133, CD44, EPCAM and CD90 as molecular markers of CSCs [11,12,13,14], and CSCs can be isolated using flow cytometry according to the expression of these specific cell surface markers, such as CD133 and CD44 [15, 16] It is still quite challenging and time-consuming to identify CSC cells by using these markers. Few studies investigated its inhibitory effect against cervical cancer stem cells
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