In Vitro Tumorigenic Assay: Colony Forming Assay for Cancer Stem Cells

Rajendran, Vijayalakshmi; Jain, Mayur Vilas

doi:10.1007/978-1-4939-7401-6_8

Cited by 117 publications

(78 citation statements)

References 11 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…On the other hand, Pin1 inhibitory effects extended to clonogenic assessment in Pin1 knockdown gastric cancer cells. Colony formation assay has been used to evaluate the survival capability of a single cancer stem cell to expand into a large clone in vitro tumorigenesis . Although the colony‐forming unit may not be the substitute of cancer stem cells with self‐renewing and differentiative capacity, we did find Pin1 expression localized in the basal cells of normal gastric mucosa, which exhibits stem‐like and proliferative properties during the regeneration of damaged tissues .…”

Section: Discussionmentioning

confidence: 75%

Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/β‐catenin oncogenic pathways

Zhang

Zheng

et al. 2019

Molecular Carcinogenesis

View full text Add to dashboard Cite

Gastric cancer is the second leading cause of cancer-related mortality and the fourth most common cancer globally. High intratumor heterogeneity of advanced gastric cancer poses great challenges to targeted therapy due to simultaneous activation of many redundant cancer-driving pathways. A central common signaling mechanism in cancer is prolinedirected phosphorylation, which is further regulated by the unique proline isomerase Pin1.Pin1 inhibition exerts anticancer activity by blocking multiple cancer-driving pathways in some cancers, but its role in gastric cancer is not fully understood. Here we detected Pin1 protein expression in 1065 gastric cancer patients and paired normal tissues using immunohistochemistry and Western blot, and then examined the effects of Pin1 overexpression, and genetic and chemical Pin1 inhibition using Pin1 short hairpin RNA or small molecule inhibitor all-trans retinoic acid (ATRA) on tumorigenesis of human gastric cancer in vitro and in vivo, followed by biochemical analyses to elucidate Pin1 regulated oncogenic pathways. We found that Pin1 was significantly overexpressed in primary and metastasized tumors, with Pin1 overexpression being correlated with advanced stage and poor prognosis. Furthermore, whereas Pin1 overexpression promoted the transformed phenotype in immortalized and nontransformed human gastric cells, either genetic or chemical Pin1 inhibition in multiple human gastric cancer cells potently suppressed cell growth, G1/S transition and colony formation in vitro, as well as tumor growth in xenograft tumor models in vivo, which were further supported by downregulation of multiple key oncoproteins in PI3K/AKT and Wnt/β-catenin signaling pathways. These results not only provide the first evidence for a critical role of Pin1 in the tumorigenesis of gastric cancer but also suggest that targeting Pin1 using ATRA or other inhibitors offers an effective new therapeutic approach for treating advanced gastric cancer.

show abstract

Section: Discussionmentioning

confidence: 75%

Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/β‐catenin oncogenic pathways

Zhang

Zheng

et al. 2019

Molecular Carcinogenesis

View full text Add to dashboard Cite

show abstract

“…Clonogenic assay is an in-vitro cell survival assay used to determine the colony formation capability of single cancer cells [49]. PFD and PFD-D-Lip were evaluated for their long-term efficacy against two different NSCLC cell lines (A549 and H4006) in a clonogenic assay.…”

Section: Clonogenic Assaymentioning

confidence: 99%

Systematic Development and Optimization of Inhalable Pirfenidone Liposomes for Non-Small Cell Lung Cancer Treatment

et al. 2020

View full text Add to dashboard Cite

Non-small cell lung cancer (NSCLC) is a global disorder, treatment options for which remain limited with resistance development by cancer cells and off-target events being major roadblocks for current therapies. The discovery of new drug molecules remains time-consuming, expensive, and prone to failure in safety/efficacy studies. Drug repurposing (i.e., investigating FDA-approved drug molecules for use against new indications) provides an opportunity to shorten the drug development cycle. In this project, we propose to repurpose pirfenidone (PFD), an anti-fibrotic drug, for NSCLC treatment by encapsulation in a cationic liposomal carrier. Liposomal formulations were optimized and evaluated for their physicochemical properties, in-vitro aerosol deposition behavior, cellular internalization capability, and therapeutic potential against NSCLC cell lines in-vitro and ex-vivo. Anti-cancer activity of PFD-loaded liposomes and molecular mechanistic efficacy was determined through colony formation (1.5- to 2-fold reduction in colony growth compared to PFD treatment in H4006, A549 cell lines, respectively), cell migration, apoptosis and angiogenesis assays. Ex-vivo studies using 3D tumor spheroid models revealed superior efficacy of PFD-loaded liposomes against NSCLC, as compared to plain PFD. Hence, the potential of inhalable liposome-loaded pirfenidone in NSCLC treatment has been established in-vitro and ex-vivo, where further studies are required to determine their efficacy through in vivo preclinical studies followed by clinical studies.

show abstract

“…Then, we investigated whether long‐term sorafenib treatment led to other features of cancer stem cells. Cancer stem cells are capable of forming large colonies through clonal expansion from a single cell, and clonogenic activity has been considered an important indicator of undifferentiation (Rajendran and Jain, ). In agreement with this notion, HepG2SF1 and Huh7SF1 cells had a higher capacity of forming colonies than their parental cells (Fig.…”

Section: Resultsmentioning

confidence: 99%

Targeting AMP‐activated kinase impacts hepatocellular cancer stem cells induced by long‐term treatment with sorafenib

et al. 2019

View full text Add to dashboard Cite

Hepatocellular carcinoma ( HCC ) is the third leading cause of cancer death worldwide. HCC treatment is hindered by the frequent emergence of chemoresistance to the multikinase inhibitor sorafenib, which has been related to the presence of cancer stem cells ( CSC s) that self‐renew and often escape therapy. The key metabolic sensor AMP ‐activated kinase ( AMPK ) has recently been recognized as a tumour growth regulator. In this study, we aimed to elucidate the role of AMPK in the development of a stem cell phenotype in HCC cells. To this end, we enriched the CSC population in HCC cell lines that showed increased expression of drug resistance ( ALDH 1A1, ABCB 1A) and stem cell ( CD 133, Nanog, Oct4, alpha fetoprotein) markers and demonstrated their stemness phenotype. These cells were refractory to sorafenib‐induced cell death. We report that sorafenib‐resistant cells had lower levels of total and phosphorylated AMPK as well as its downstream substrate, ACC , compared with the parental cells. Interestingly, AMPK knockdown with si RNA or inhibition with dorsomorphin increased the expression of stem cell markers in parental cells and blocked sorafenib‐induced cell death. Conversely, the upregulation of AMPK , either by transfection or by pharmacological activation with A‐769662, decreased the expression of ALDH 1A1, ABCB 1A, CD 133, Nanog, Oct4, and alpha fetoprotein, and restored sensitivity to sorafenib. Analysis of the underlying mechanism points to hypoxia‐inducible factor HIF ‐1α as a regulator of stemness. In vivo studies in a xenograft mouse model demonstrated that stem‐like cells have greater tumourigenic capacity. AMPK activation reduced xenograft tumour growth and decreased the expression of stem cell markers. Taken together, these results indicate that AMPK may serve as a novel target to overcome chemoresistance in HCC .

show abstract

In Vitro Tumorigenic Assay: Colony Forming Assay for Cancer Stem Cells

Cited by 117 publications

References 11 publications

Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/β‐catenin oncogenic pathways

Pin1 inhibition potently suppresses gastric cancer growth and blocks PI3K/AKT and Wnt/β‐catenin oncogenic pathways

Systematic Development and Optimization of Inhalable Pirfenidone Liposomes for Non-Small Cell Lung Cancer Treatment

Targeting AMP‐activated kinase impacts hepatocellular cancer stem cells induced by long‐term treatment with sorafenib

Contact Info

Product

Resources

About