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Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

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HCI2509 induces apoptotic cell death. (A, B) Cell viability and caspase activation at 0, 24, 48, and 72 hours in (A) AN3CA and (B) KLE cells treated with 3X EC50 HCI2509. Measurements were normalized to their respective vehicle (0.3% DMSO) sample at the appropriate time point. (C, D) Fluorescence microscopy images of (C) AN3CA and (D) KLE cell lines after exposure to either vehicle or 3X EC50 HCI2509 and then stained with TUNEL for apoptotic nuclei (green), DAPI for nuclei (blue), and phalloidin for actin (red). HCI2509 treatment induced apoptosis with apoptotic cells marked with (*).
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Fig4: HCI2509 induces apoptotic cell death. (A, B) Cell viability and caspase activation at 0, 24, 48, and 72 hours in (A) AN3CA and (B) KLE cells treated with 3X EC50 HCI2509. Measurements were normalized to their respective vehicle (0.3% DMSO) sample at the appropriate time point. (C, D) Fluorescence microscopy images of (C) AN3CA and (D) KLE cell lines after exposure to either vehicle or 3X EC50 HCI2509 and then stained with TUNEL for apoptotic nuclei (green), DAPI for nuclei (blue), and phalloidin for actin (red). HCI2509 treatment induced apoptosis with apoptotic cells marked with (*).

Mentions: In addition to cell cycle disruption, we investigated the mechanism causing negative cell doubling in both AN3CA and KLE cells. We hypothesized that HCI2509 treatment may cause apoptotic cell death and therefore tested both cell lines for caspase 3/7 activation. Caspase activity was assayed in parallel with cell viability using 3X the EC50 and comparing to vehicle control. Viability and caspase activation were assessed over a time-course of 72 hours in both cell lines. Interestingly, in the context of HCI2509 treatment AN3CA showed decreased cell viability and caspase activity over the course of 48 hours with increased caspase activation occurring at 72 hours (Figure 4A). The decrease in caspase activation during the first 48 hours of treatment is likely due to a decreased number of cells/well due to cytostatsis relative to vehicle. HCI2509-treated KLE cells showed a concomitant increase in caspase activity and decrease in cell viability over 72 hours (Figure 4B). These data suggest an initial cytostasis which is followed by apoptotic cell death induced after 48 hours. We next confirmed apoptotic cell death using fluorescent TUNEL staining. AN3CA and KLE cells were treated with either vehicle or 3X EC50 HCI2509 for 72 hours and then assayed for TUNEL staining. Both cell lines showed decreased cell density and the presence of apoptotic cells with HCI2509 treatment, while vehicle treated cells appeared healthy and well spread on the coverslip (Figure 4C, D, Additional file 3: Figure S4A, S4B). Internal controls for the TUNEL assay are reported in Additional file 3: Figure S4C. These results confirmed apoptotic cell death induced by HCI2509 treatment.Figure 4


Reversible inhibition of lysine specific demethylase 1 is a novel anti-tumor strategy for poorly differentiated endometrial carcinoma.

Theisen ER, Gajiwala S, Bearss J, Sorna V, Sharma S, Janat-Amsbury M - BMC Cancer (2014)

HCI2509 induces apoptotic cell death. (A, B) Cell viability and caspase activation at 0, 24, 48, and 72 hours in (A) AN3CA and (B) KLE cells treated with 3X EC50 HCI2509. Measurements were normalized to their respective vehicle (0.3% DMSO) sample at the appropriate time point. (C, D) Fluorescence microscopy images of (C) AN3CA and (D) KLE cell lines after exposure to either vehicle or 3X EC50 HCI2509 and then stained with TUNEL for apoptotic nuclei (green), DAPI for nuclei (blue), and phalloidin for actin (red). HCI2509 treatment induced apoptosis with apoptotic cells marked with (*).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4197342&req=5

Fig4: HCI2509 induces apoptotic cell death. (A, B) Cell viability and caspase activation at 0, 24, 48, and 72 hours in (A) AN3CA and (B) KLE cells treated with 3X EC50 HCI2509. Measurements were normalized to their respective vehicle (0.3% DMSO) sample at the appropriate time point. (C, D) Fluorescence microscopy images of (C) AN3CA and (D) KLE cell lines after exposure to either vehicle or 3X EC50 HCI2509 and then stained with TUNEL for apoptotic nuclei (green), DAPI for nuclei (blue), and phalloidin for actin (red). HCI2509 treatment induced apoptosis with apoptotic cells marked with (*).
Mentions: In addition to cell cycle disruption, we investigated the mechanism causing negative cell doubling in both AN3CA and KLE cells. We hypothesized that HCI2509 treatment may cause apoptotic cell death and therefore tested both cell lines for caspase 3/7 activation. Caspase activity was assayed in parallel with cell viability using 3X the EC50 and comparing to vehicle control. Viability and caspase activation were assessed over a time-course of 72 hours in both cell lines. Interestingly, in the context of HCI2509 treatment AN3CA showed decreased cell viability and caspase activity over the course of 48 hours with increased caspase activation occurring at 72 hours (Figure 4A). The decrease in caspase activation during the first 48 hours of treatment is likely due to a decreased number of cells/well due to cytostatsis relative to vehicle. HCI2509-treated KLE cells showed a concomitant increase in caspase activity and decrease in cell viability over 72 hours (Figure 4B). These data suggest an initial cytostasis which is followed by apoptotic cell death induced after 48 hours. We next confirmed apoptotic cell death using fluorescent TUNEL staining. AN3CA and KLE cells were treated with either vehicle or 3X EC50 HCI2509 for 72 hours and then assayed for TUNEL staining. Both cell lines showed decreased cell density and the presence of apoptotic cells with HCI2509 treatment, while vehicle treated cells appeared healthy and well spread on the coverslip (Figure 4C, D, Additional file 3: Figure S4A, S4B). Internal controls for the TUNEL assay are reported in Additional file 3: Figure S4C. These results confirmed apoptotic cell death induced by HCI2509 treatment.Figure 4

Bottom Line: These effects were largely dose-dependent.Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen.Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmaceutics and Pharmaceutical Chemistry, College of Pharmacy, University of Utah, Salt Lake City, UT, USA. margit.janat-amsbury@hsc.utah.edu.

ABSTRACT

Background: Endometrial cancer is the most common gynecologic malignancy. Type II endometrial carcinoma is often poorly differentiated and patients diagnosed with Type II disease (~11%) are disproportionately represented in annual endometrial cancer deaths (48%). Recent genomic studies highlight mutations in chromatin regulators as drivers in Type II endometrial carcinoma tumorigenesis, suggesting the use of epigenetic targeted therapies could provide clinical benefit to these patients. We investigated the anti-tumor efficacy of the LSD1 inhibitor HCI2509 in two poorly differentiated Type II endometrial cancer cell lines AN3CA and KLE.

Methods: The effects of HCI2509 on viability, proliferation, anchorage-independent growth, global histone methylation, LSD1 target gene induction, cell cycle, caspase activation and TUNEL were assayed. KLE cells were used in an orthotopic xenograft model to assess the anti-tumor activity of HCI2509.

Results: Both AN3CA and KLE cells were sensitive to HCI2509 treatment with IC50s near 500 nM for cell viability. Inhibition of LSD1 with HCI2509 caused decreased proliferation and anchorage independent growth in soft agar, elevated global histone methylation, and perturbed the cell cycle in both cell lines. These effects were largely dose-dependent. HCI2509 treatment also caused apoptotic cell death. Orthotopic implantation of KLE cells resulted in slow-growing and diffuse tumors throughout the abdomen. Tumor burden was distributed log-normally. Treatment with HCI2509 resulted 5/9 tumor regressions such that treatment and regressions were significantly associated (p=0.034).

Conclusions: Our findings demonstrate the anti-cancer properties of the LSD1 inhibitor HCI2509 on poorly differentiated endometrial carcinoma cell lines, AN3CA and KLE. HCI2509 showed single-agent efficacy in orthotopic xenograft studies. Continued studies are needed to preclinically validate LSD1 inhibition as a therapeutic strategy for endometrial carcinoma.

Show MeSH
Related in: MedlinePlus