Limits...
Involvement of Seladin-1 in goniothalamin-induced apoptosis in urinary bladder cancer cells.

Yen HK, Fauzi AR, Din LB, McKelvey-Martin VJ, Meng CK, Inayat-Hussain SH, Rajab NF - BMC Complement Altern Med (2014)

Bottom Line: This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells.This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells.The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Science Programme, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. nfadilah@medic.ukm.my.

ABSTRACT

Background: Selective Alzheimer Disease Indicator-1 (or Seladin-1) is a multifunctional protein first discovered by downregulation of its expression in Alzheimer's disease. Interestingly, the expression of this protein is upregulated in several cancers, including primary bladder cancer. However, its role in cancer formation has yet to be discovered. Goniothalamin is a natural product that has been demonstrated to induce apoptosis in various cancer cell lines. In this study, we have elucidated the role of Seladin-1 in goniothalamin-induced cytotoxicity towards human urinary bladder cancer cell line RT4.

Methods: The cytotoxicity of goniothalamin in human urinary bladder cancer cell line RT4 was assessed using MTT assay and the mode of cell death was determined by Annexin V-FITC/PI labeling assay. Finally, the expression of Seladin-1 protein in goniothalamin-treated RT4 cells was determined by Western blot.

Results: MTT assay showed that the cytotoxicity of goniothalamin on RT4 cells was concentration and time dependent with IC50 values of 61 μM (24 hr), 38 μM (48 hr) and 31 μM for 72 hr, respectively. Cell death induced was confirmed through apoptosis; as assessed using the Annexin V-FITC/PI labeling assay. Furthermore, the involvement of Seladin-1 in goniothalamin-induced apoptosis was evidenced through the cleavage of 60 kDa protein to 40 kDa and 20 kDa. This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells.

Conclusion: This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells. The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.

Show MeSH

Related in: MedlinePlus

Flow cytometry profile of RT4 cells treated by goniohtalamin (GTN) at 50, 61.3 (IC50), and 100 μM, etoposide IC50for 24 hrs., a) Untreated cells (VC) b) GTN 50 μM; c) IC50GTN d) 100 μM GTN and e) etoposide IC50.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC4150971&req=5

Fig3: Flow cytometry profile of RT4 cells treated by goniohtalamin (GTN) at 50, 61.3 (IC50), and 100 μM, etoposide IC50for 24 hrs., a) Untreated cells (VC) b) GTN 50 μM; c) IC50GTN d) 100 μM GTN and e) etoposide IC50.

Mentions: Goniothalamin was known to induce cell death with mainly through apoptosis at various cell lines [17–23]. In this study mode of cell death or determination of apoptosis event induced by goniothalamin on bladder cancer cell line RT4 was assessed by using Annexin V-FITC/PI Labeling assay as described previously [25], at the concentration of 50, 61 (IC50) &100 μM of goniothalamin following 24 hr treatment. Three concentration of goniothalamin was chosen in order to understand the concentration dependent cell death event induced by goniothalamin. By using Annexin V-FITC/PI staining, apoptosis and necrosis event was quantified by flow cytometry as shown in Figure 3. The cytogram quadrant of annexin V staining positive represent apoptotic event and PI staining positive represent necrotic event. Quadrant 1 (Q1) is the region of necrotic event where annexin V staining negative and PI staining positive. Q2 is late apoptosis event where both annexin V and PI staining positive, Q4 is early apoptosis event with cells stained with annexin V alone and Q3 represent event of negative staining for both annexin V and PI. Based on the result, there was an increased in the apoptosis event with the increase of goniothalamin concentration. At 50 μM, 53.6% of the cells are apoptotic, whereas 63.8% are apoptotic at 61μM treatment and 70.5% at 100 μM, respectively. However, etoposide showed only 25.1% of apoptosis event. Only small percentages of cells were necrotic. Vehicle control or untreated cell was also assessed to ensure the viability of cell for each experiment, majority all the event fall into Q 3 which was 81.9% of cell stained negative for both stain. Statistical analysis showed significant apoptosis event compare to untreated cells with p < 0.05, with no significant difference for necrotic events.Figure 3


Involvement of Seladin-1 in goniothalamin-induced apoptosis in urinary bladder cancer cells.

Yen HK, Fauzi AR, Din LB, McKelvey-Martin VJ, Meng CK, Inayat-Hussain SH, Rajab NF - BMC Complement Altern Med (2014)

Flow cytometry profile of RT4 cells treated by goniohtalamin (GTN) at 50, 61.3 (IC50), and 100 μM, etoposide IC50for 24 hrs., a) Untreated cells (VC) b) GTN 50 μM; c) IC50GTN d) 100 μM GTN and e) etoposide IC50.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4150971&req=5

Fig3: Flow cytometry profile of RT4 cells treated by goniohtalamin (GTN) at 50, 61.3 (IC50), and 100 μM, etoposide IC50for 24 hrs., a) Untreated cells (VC) b) GTN 50 μM; c) IC50GTN d) 100 μM GTN and e) etoposide IC50.
Mentions: Goniothalamin was known to induce cell death with mainly through apoptosis at various cell lines [17–23]. In this study mode of cell death or determination of apoptosis event induced by goniothalamin on bladder cancer cell line RT4 was assessed by using Annexin V-FITC/PI Labeling assay as described previously [25], at the concentration of 50, 61 (IC50) &100 μM of goniothalamin following 24 hr treatment. Three concentration of goniothalamin was chosen in order to understand the concentration dependent cell death event induced by goniothalamin. By using Annexin V-FITC/PI staining, apoptosis and necrosis event was quantified by flow cytometry as shown in Figure 3. The cytogram quadrant of annexin V staining positive represent apoptotic event and PI staining positive represent necrotic event. Quadrant 1 (Q1) is the region of necrotic event where annexin V staining negative and PI staining positive. Q2 is late apoptosis event where both annexin V and PI staining positive, Q4 is early apoptosis event with cells stained with annexin V alone and Q3 represent event of negative staining for both annexin V and PI. Based on the result, there was an increased in the apoptosis event with the increase of goniothalamin concentration. At 50 μM, 53.6% of the cells are apoptotic, whereas 63.8% are apoptotic at 61μM treatment and 70.5% at 100 μM, respectively. However, etoposide showed only 25.1% of apoptosis event. Only small percentages of cells were necrotic. Vehicle control or untreated cell was also assessed to ensure the viability of cell for each experiment, majority all the event fall into Q 3 which was 81.9% of cell stained negative for both stain. Statistical analysis showed significant apoptosis event compare to untreated cells with p < 0.05, with no significant difference for necrotic events.Figure 3

Bottom Line: This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells.This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells.The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Science Programme, Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Jalan Raja Muda Abdul Aziz, 50300 Kuala Lumpur, Malaysia. nfadilah@medic.ukm.my.

ABSTRACT

Background: Selective Alzheimer Disease Indicator-1 (or Seladin-1) is a multifunctional protein first discovered by downregulation of its expression in Alzheimer's disease. Interestingly, the expression of this protein is upregulated in several cancers, including primary bladder cancer. However, its role in cancer formation has yet to be discovered. Goniothalamin is a natural product that has been demonstrated to induce apoptosis in various cancer cell lines. In this study, we have elucidated the role of Seladin-1 in goniothalamin-induced cytotoxicity towards human urinary bladder cancer cell line RT4.

Methods: The cytotoxicity of goniothalamin in human urinary bladder cancer cell line RT4 was assessed using MTT assay and the mode of cell death was determined by Annexin V-FITC/PI labeling assay. Finally, the expression of Seladin-1 protein in goniothalamin-treated RT4 cells was determined by Western blot.

Results: MTT assay showed that the cytotoxicity of goniothalamin on RT4 cells was concentration and time dependent with IC50 values of 61 μM (24 hr), 38 μM (48 hr) and 31 μM for 72 hr, respectively. Cell death induced was confirmed through apoptosis; as assessed using the Annexin V-FITC/PI labeling assay. Furthermore, the involvement of Seladin-1 in goniothalamin-induced apoptosis was evidenced through the cleavage of 60 kDa protein to 40 kDa and 20 kDa. This was followed by a gradual increase of 20 kDa fragment suggesting the involvement of Seladin-1 in goniothalamin-induced apoptosis on RT4 cells.

Conclusion: This study demonstrates that goniothalamin induce cytotoxicity and apoptosis on RT4 cells. The involvement of Seladin-1 in goniothalamin-induced apoptosis further suggested that Seladin-1 may play a role in the formation of primary bladder cancer.

Show MeSH
Related in: MedlinePlus