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Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.

Liu Y, Song N, Ren K, Meng S, Xie Y, Long Q, Chen X, Zhao X - PLoS ONE (2013)

Bottom Line: Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05).RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05).TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology & Obstetrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.

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Revivification of RhoB gene and apoptosis of ovary carcinoma cells.Flow cytometric analysis (FCM) together with fluorescence microscopy was adopted to assess apoptosis of ovary carcinoma cells after treated with TSA. Top and middle panels: when cells were treated for 10 h with 0 µmol/L (control), 0.05 µmol/L, 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L and 1.0 µmol/L of TSA, the apoptosis rate revealed by FCM was 9.5% (A), 26.9% (B), 28% (C), 41% (D), 45.9% (E) and 66.9% (F) respectively. Bottom panel: PI stained fluorescence photomicrographs of ovary carcinoma cells after treated with TSA. Cells were treated for 10h with 0 µmol/L (G, control), 0.1 µmol/L and 0.5 µmol/L of TSA respectively, both 0.1 µmol/L (H) and 0.5µmol/L (I) of TSA could result in cellular morphological changes characterized as apoptosis: a brightly red-fluorescent condensed nuclei (intact or fragmented), reduction of cell volume, and apoptotic bodies.
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pone-0078417-g005: Revivification of RhoB gene and apoptosis of ovary carcinoma cells.Flow cytometric analysis (FCM) together with fluorescence microscopy was adopted to assess apoptosis of ovary carcinoma cells after treated with TSA. Top and middle panels: when cells were treated for 10 h with 0 µmol/L (control), 0.05 µmol/L, 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L and 1.0 µmol/L of TSA, the apoptosis rate revealed by FCM was 9.5% (A), 26.9% (B), 28% (C), 41% (D), 45.9% (E) and 66.9% (F) respectively. Bottom panel: PI stained fluorescence photomicrographs of ovary carcinoma cells after treated with TSA. Cells were treated for 10h with 0 µmol/L (G, control), 0.1 µmol/L and 0.5 µmol/L of TSA respectively, both 0.1 µmol/L (H) and 0.5µmol/L (I) of TSA could result in cellular morphological changes characterized as apoptosis: a brightly red-fluorescent condensed nuclei (intact or fragmented), reduction of cell volume, and apoptotic bodies.

Mentions: With the treatment by TSA for 10 h, the concentration leading to a 50% decrease in cell number (IC50) was about 0.5μmol/L. Based on the result, SKOV3 and A2780 cells were exposed to increase doses of TSA (0.05-1.0 µmol/L) for 10 h, and all the cells were harvested to measure apoptosis level by FCM. Even low dose of 0.05µmol/L TSA could cause 26.9 % apoptosis on the cells (p<0.05), and high dose (0.1, 0.25, 0.5 and 1.0µmol/L) of TSA could cause 28 %, 41%, 45.9% and 66.9% apoptosis respectively (Figure 5A-F). These data indicated that TSA could mediate apoptosis of ovarian tumor cells in a concentration-dependent manner. PI stained fluorescence photomicrography for the cells revealed morphological changes characterized as apoptosis after treated with TSA: a brightly red-fluorescent condensed nuclei (fragmented or intact), reduction of cell volume, or apoptotic bodies (Figure 5G-I). Results obtained in flow cytometry are strongly correlated with morphological changes in PI-stained fluorescence photomicrography.


Expression loss and revivification of RhoB gene in ovary carcinoma carcinogenesis and development.

Liu Y, Song N, Ren K, Meng S, Xie Y, Long Q, Chen X, Zhao X - PLoS ONE (2013)

Revivification of RhoB gene and apoptosis of ovary carcinoma cells.Flow cytometric analysis (FCM) together with fluorescence microscopy was adopted to assess apoptosis of ovary carcinoma cells after treated with TSA. Top and middle panels: when cells were treated for 10 h with 0 µmol/L (control), 0.05 µmol/L, 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L and 1.0 µmol/L of TSA, the apoptosis rate revealed by FCM was 9.5% (A), 26.9% (B), 28% (C), 41% (D), 45.9% (E) and 66.9% (F) respectively. Bottom panel: PI stained fluorescence photomicrographs of ovary carcinoma cells after treated with TSA. Cells were treated for 10h with 0 µmol/L (G, control), 0.1 µmol/L and 0.5 µmol/L of TSA respectively, both 0.1 µmol/L (H) and 0.5µmol/L (I) of TSA could result in cellular morphological changes characterized as apoptosis: a brightly red-fluorescent condensed nuclei (intact or fragmented), reduction of cell volume, and apoptotic bodies.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3815309&req=5

pone-0078417-g005: Revivification of RhoB gene and apoptosis of ovary carcinoma cells.Flow cytometric analysis (FCM) together with fluorescence microscopy was adopted to assess apoptosis of ovary carcinoma cells after treated with TSA. Top and middle panels: when cells were treated for 10 h with 0 µmol/L (control), 0.05 µmol/L, 0.1 µmol/L, 0.25 µmol/L, 0.5 µmol/L and 1.0 µmol/L of TSA, the apoptosis rate revealed by FCM was 9.5% (A), 26.9% (B), 28% (C), 41% (D), 45.9% (E) and 66.9% (F) respectively. Bottom panel: PI stained fluorescence photomicrographs of ovary carcinoma cells after treated with TSA. Cells were treated for 10h with 0 µmol/L (G, control), 0.1 µmol/L and 0.5 µmol/L of TSA respectively, both 0.1 µmol/L (H) and 0.5µmol/L (I) of TSA could result in cellular morphological changes characterized as apoptosis: a brightly red-fluorescent condensed nuclei (intact or fragmented), reduction of cell volume, and apoptotic bodies.
Mentions: With the treatment by TSA for 10 h, the concentration leading to a 50% decrease in cell number (IC50) was about 0.5μmol/L. Based on the result, SKOV3 and A2780 cells were exposed to increase doses of TSA (0.05-1.0 µmol/L) for 10 h, and all the cells were harvested to measure apoptosis level by FCM. Even low dose of 0.05µmol/L TSA could cause 26.9 % apoptosis on the cells (p<0.05), and high dose (0.1, 0.25, 0.5 and 1.0µmol/L) of TSA could cause 28 %, 41%, 45.9% and 66.9% apoptosis respectively (Figure 5A-F). These data indicated that TSA could mediate apoptosis of ovarian tumor cells in a concentration-dependent manner. PI stained fluorescence photomicrography for the cells revealed morphological changes characterized as apoptosis after treated with TSA: a brightly red-fluorescent condensed nuclei (fragmented or intact), reduction of cell volume, or apoptotic bodies (Figure 5G-I). Results obtained in flow cytometry are strongly correlated with morphological changes in PI-stained fluorescence photomicrography.

Bottom Line: Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05).RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05).TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't.

View Article: PubMed Central - PubMed

Affiliation: Department of Gynecology & Obstetrics, West China Second Hospital, Sichuan University, Chengdu, Sichuan, People's Republic of China.

ABSTRACT
RhoB, a member of small GTPases belonging to the Ras protein superfamily, might have a suppressive activity in cancer progression. Here, expression of RhoB gene was evaluated in human benign, borderline and malignant ovary tumors by immunostaining, with normal ovary tissue as control. Malignant tumors were assessed according to Federation Internationale de Gynecologie Obstetrique (FIGO) guidelines and classified in stage I-IV. Revivification of RhoB gene was investigated by analyzing the effect of histone deacetylase (HDAC) inhibitor trichostatin (TSA) and methyltransferase inhibitor 5-azacytidine (5-Aza) on ovarian cancer cells via RT-PCR and western blot. Apoptosis of ovary cancer cells was detected using flowcytometry and fluorescence microscopy. Subsequently, RhoB expression is detected in normal ovary epithelium, borderline tumors, and decreases significantly or lost in the majority of ovarian cancer specimen (P<0.05). RhoB expression decreases significantly from stage II (71.4%) to stage III (43.5%) to stage IV (18.2%, P<0.05). TSA can both significantly revive the RhoB gene and mediate apoptosis of ovarian cancer cells, but 5-Aza couldn't. Interference into Revivification of RhoB gene results in reduction of ovary carcinoma cell apoptosis. It is proposed that loss of RhoB expression occurs frequently in ovary carcinogenesis and progression and its expression could be regulated by histone deacetylation but not by promoter hypermethylation, which may serve as a prospective gene treatment target for the patients with ovarian malignancy not responding to standard therapies.

Show MeSH
Related in: MedlinePlus