<p>Phase contrast images of the three MDA-MB-231 cell lines. Image (A) shows MDA-MB-231P ce... more <p>Phase contrast images of the three MDA-MB-231 cell lines. Image (A) shows MDA-MB-231P cells grown in DMEM 0 mM glucose images; (B–C) the same cells grown in DMEM with different glucose concentrations: 5 mM and 25 mM. Image (D) represents MDA-MB-231BO cells growing in DMEM 0 mM glucose; images (E–F) are for the same cells grown in the other glucose concentrations. Neither MDA-MB-231P nor MDA-MB-231BO cells were morphologically changed by the various changes in glucose concentration. The MDA-MB-231BR cells grown in the absence of glucose for 24 hrs lost their extended protrusions (G), became rounded and maintained their attachment to the culture plate; images (H and I) show how the shape of the cells was restored upon the increase in glucose concentration. Scale bars = 100 µm. Breast cancer cells formed fewer colonies and had reduced proliferation under hypoglycemic conditions. (B) Graph represents the ratio of the average number of colonies counted in four independent experiments. The y-axis represents the normalized number of colonies counted in the hypoglycemia exposed wells versus the control formed by the cells. MDA-MB-231BR cells produced the least colonies among the three cell lines when exposed to hypoglycemia and this was confirmed by running the non-parametric Kruskal Wallis test H = 15.66 and p = 0.0013. (C) The graph represents cell growth as the percentage of Alamarblue reduced by the cells under hypo/hyperglycemia. There was a significant decrease in the reduced Alamarblue when the cells were grown in hypoglycemia for 24 hrs (MDA-MB-231P, MDA-MB-231BO, and MDA-MB-231BR) Results are shown as mean ± SD and were statistically analyzed by a two-tailed student’s t test (n = 3).</p
<p>(A) Integrin β4 was weakly expressed, or undetectable in the MDA-MB-231BO and MDA-MB-231... more <p>(A) Integrin β4 was weakly expressed, or undetectable in the MDA-MB-231BO and MDA-MB-231BR cells respectively however, it was expressed in all glycemic conditions in MDA-MB-231P cells which was significantly higher than the variants cells p>0.001 (graph on the right). Integrin αv was down regulated in MDA-MB-231BO and not detected when cells were exposed to DMEM 0 mM glucose (B0) (graph on far right). Integrin α-V was significantly decreased under hypoglycemia only in MDA-MB-231P cells p = 0.001. Integrin α5 was expressed in all the three cell lines and was down regulated in the absence of glucose in all the cells in study (bottom graph on the right and lower left panel). Integrin β3 was not detectable in the MDA-MB-231BO cells, and barely detectable in the MDA-MB-231P cells. It was expressed in detectable levels in MDA-MB-231BR cells and was slightly down regulated in reduced glucose. Error bars represent ± standard deviation. Note: β-Actin was used in all the blots as a protein loading control.</p
<p>(A) Cyclohexamide treatment for a period of 24 hrs depleted VEGFR2 protein from all thre... more <p>(A) Cyclohexamide treatment for a period of 24 hrs depleted VEGFR2 protein from all three cell lines. The blot in this panel demonstrates that cyclohexamide depleted VEGFR2 from the MDA-MB-231BO cells (regularly expressing the highest level of VEGFR2 among the three cell lines) whether they were hypoglycemic or hyperglycemic (lane 1 and 3). The control cyclohexamide negative samples –glu −CHX and +glu -CHX showed detectable VEGFR2 expression (lane 2 and 4). (B) VEGFR2 was completely depleted from the three cell lines when grown in hypoglycemia in the presence of CHX (lane 2). The samples in lanes 3 and 5 are cells exposed to hypoglycemia for 24 hrs without CHX and glucose was restored for 1 hr (lane 3) or 3h (lane 5), respectively. Comparing the former two lanes (3 and 5) shows clearly that the VEGFR2 band was increasing in molecular weight as the time of glucose exposure increased; this is very prominent in the middle (MDA-MB-231BO) and bottom blots (MDA-MB-231BR). The samples in lane 4 and 6 were glucose restored for 1 hr and 3 hrs respectively with CHX added at the time of glucose restoration. After 3 hr of CHX and glucose restoration of MDA-MB-231P cells, the VEGFR2 band was not detectable (lane 6- P), however the MDA-MB-231BO and MDA-MB-231BR cells (lane 6 BO, lane 6 BR) expressed a detectable VEGFR2 band. Note: β-Actin was used in all the blots as a protein loading control. (C) Stability of NRP-1 expression under low glucose and CHX treatment. NRP-1 was detected in the same samples as in the previous panel (B) and it did not show a profound decrease in its expression in both MDA-MB-231BO and MDA-MB-231BR. However in MDA-MB-231P samples the NRP-1 band pattern was changed after 24 hrs of hypoglycemia in the presence of CHX (lane 2). The NRP-1 band was also decreased in the glucose-restored samples with CHX (added at the time of restoration) (lanes 6, 8, and 10). (D) The blot shows that in MDA-MB-231BR cells the NRP-1 protein was resistant to CHX treatment for up to 72 hrs of exposure. Note: β-Actin was used in all the blots as a protein loading control.</p
<p>(A) Western blots show the difference in VEGFR2 and phospho-VEGFR2 in MDA-MB-231 parenta... more <p>(A) Western blots show the difference in VEGFR2 and phospho-VEGFR2 in MDA-MB-231 parental (P), bone metastatic (BO) and brain metastatic (BR) variants growing in DMEM containing 25 mM glucose. (B) Graph represents the mean of four independent ELISA experiments measuring the total VEGF-A secreted in the conditioned media, (error bars represent ± standard deviation). Statistical analysis showed no significant difference in the amount of VEGF-A secreted when the MDA-MB-231P and MDA-MB-231BO cells were grown under the different glucose concentrations. However, MDA-MB-231BR cells produced significantly higher levels of VEGF-A (p = 0.012) when grown in DMEM containing 25 mM of glucose compared to 0 mM of glucose. Figure abbreviations P: MDA-MB-231 Parental, BO: MDA-MB-231Bone and BR: MDA-MB-231Brain; cell lines were grown in DMEM 0, 5 and 25 mM glucose for 24 hrs without FBS. (C) VEGFR2 expression in MDA-MB-231P, MDA-MB-231BO and MDA-MB-231BR cells. The 147 kDa band was the dominant band expressed at 0 mM glucose. The regular double bands (200 kDa and 230 kDa) were detected in all the cells at 25 mM glucose. The cells expressed the bands differentially when placed in 5 mM glucose media. The MDA-MB-231P cells produced the 200 and 230 kDa forms; the MDA-MB-231BO cells produced the 147 and 200 kDa forms while the MDA-MB-231BR cells did not express any of the mature glycosylated bands at 5 mM glucose concentration. The bottom blot shows the expression of VEGFR1 was constant and did not change among the cell lines nor under the different glucose conditions. (D) Blot shows MDA-MB-231BR cells produced higher levels of GLUT-1 protein than the other cell lines. GRP78 had the same expression pattern among the different cells and its expression had an inverse relationship with glucose concentration. Note: β-Actin was used in all the blots as protein loading control.</p
<p> <b>hrs.</b> (A) The graph (left) shows real time PCR results for the positi... more <p> <b>hrs.</b> (A) The graph (left) shows real time PCR results for the positive expression of E-cadherin in MCF-7 and BT-474 cell lines; the grey and black bars represent two repeated independent experiments. However, MDA-MB-231P and its two variant cell lines did not express detectable E-cadherin message or protein (right); (B) Representative blots (left) and quantification (right) shows that MDA-MB-231P and MDA-MB-231BO cells expressed higher levels of N-cadherin compared with the MDA-MB-231BR cells, and that N-cadherin in the former cells was significantly reduced under hypoglycemia. γ, β and α-Catenins were all reduced under hypoglycemic conditions and their expression was significantly decreased in the two MDA-MB-231 variant cells only. Error bars represent ± standard deviation. Note: β-Actin was used in all the blots as a protein loading control.</p
Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism ... more Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism by which glycemic load can exert its action on tumor metastasis is under investigated. In this study we showed that glycemic microenvironment alters the expression of three classes of proteins, VEGF and its receptors, cell to cell, and cell to extracellular matrix (ECM) adhesion proteins in MDA-MB-231 parental cells and its two metastatic variants to the bone and brain (MDA-MB-231BO and MDA-MB-231BR, respectively). Using western blotting, we showed that VEGFR2 levels were higher in these variant cells and persisted in the cells under extreme hypoglycemia. Hypoglycemia did not alter VEGFR2 expression per se but rather suppressed its posttranslational glycosylation. This was reversed rapidly upon the restoration of glucose, and cyclohexamide (CHX) treatment demonstrated that this deglycosylated VEGFR2 was not a product of de-novo protein synthesis. VEGFR2 co-receptor Neuropilin-1 was up-regulated four-fold in all MDA-MB-231 cells (parental and two variants) compared to VEGFR2 expression, and was also susceptible to glycemic changes but resistant to CHX treatment for up to 72 hrs. Hypoglycemia also resulted in a significant decrease in specific catenin, cadherin, and integrin proteins, as well as cellular proliferation and colony forming ability. However, MDA-MB-231BR cells showed a unique sensitivity to hypo/ hyperglycemia in terms of morphological changes, colony formation ability, integrin b3 expression and secreted VEGF levels. In conclusion, this study can be translated clinically to provide insight into breast cancer cell responses to glycemic levels relevant for our understanding of the interaction between diabetes and cancer.
2019 1st International Conference on Advances in Science, Engineering and Robotics Technology (ICASERT)
With the ever increasing population of the world, diseases and their possibilities are also incre... more With the ever increasing population of the world, diseases and their possibilities are also increasing at an alarming rate. As time passes by, diagnosing diseases and providing appropriate treatment at the right time has become quite a challenge. Heart diseases have been a major cause of death worldwide. Therefore, this research has been focused on finding an efficient way to predict the chances of a heart failure and accordingly, recommend appropriate medicines to aid cardiologists in quicker decision making. The research includes finding the correlations or associations between the various medical profiles of the patients by utilizing the standard techniques of exploratory analysis and hence using the attributes suitably to predict the chances of a heart failure, as well as the medicine recommendations. A comparative study has also been included which shows the various attained accuracy rates of different machine learning algorithms including - Logistic Regression, Naïve Bayes, Decision Tree, Linear Support Vector Classifier, Random Forest, and Gradient Boosting Classifier. The Apache Spark framework has been used in order to make the system capable of handling big data.
<p>Phase contrast images of the three MDA-MB-231 cell lines. Image (A) shows MDA-MB-231P ce... more <p>Phase contrast images of the three MDA-MB-231 cell lines. Image (A) shows MDA-MB-231P cells grown in DMEM 0 mM glucose images; (B–C) the same cells grown in DMEM with different glucose concentrations: 5 mM and 25 mM. Image (D) represents MDA-MB-231BO cells growing in DMEM 0 mM glucose; images (E–F) are for the same cells grown in the other glucose concentrations. Neither MDA-MB-231P nor MDA-MB-231BO cells were morphologically changed by the various changes in glucose concentration. The MDA-MB-231BR cells grown in the absence of glucose for 24 hrs lost their extended protrusions (G), became rounded and maintained their attachment to the culture plate; images (H and I) show how the shape of the cells was restored upon the increase in glucose concentration. Scale bars = 100 µm. Breast cancer cells formed fewer colonies and had reduced proliferation under hypoglycemic conditions. (B) Graph represents the ratio of the average number of colonies counted in four independent experiments. The y-axis represents the normalized number of colonies counted in the hypoglycemia exposed wells versus the control formed by the cells. MDA-MB-231BR cells produced the least colonies among the three cell lines when exposed to hypoglycemia and this was confirmed by running the non-parametric Kruskal Wallis test H = 15.66 and p = 0.0013. (C) The graph represents cell growth as the percentage of Alamarblue reduced by the cells under hypo/hyperglycemia. There was a significant decrease in the reduced Alamarblue when the cells were grown in hypoglycemia for 24 hrs (MDA-MB-231P, MDA-MB-231BO, and MDA-MB-231BR) Results are shown as mean ± SD and were statistically analyzed by a two-tailed student’s t test (n = 3).</p
<p>(A) Integrin β4 was weakly expressed, or undetectable in the MDA-MB-231BO and MDA-MB-231... more <p>(A) Integrin β4 was weakly expressed, or undetectable in the MDA-MB-231BO and MDA-MB-231BR cells respectively however, it was expressed in all glycemic conditions in MDA-MB-231P cells which was significantly higher than the variants cells p>0.001 (graph on the right). Integrin αv was down regulated in MDA-MB-231BO and not detected when cells were exposed to DMEM 0 mM glucose (B0) (graph on far right). Integrin α-V was significantly decreased under hypoglycemia only in MDA-MB-231P cells p = 0.001. Integrin α5 was expressed in all the three cell lines and was down regulated in the absence of glucose in all the cells in study (bottom graph on the right and lower left panel). Integrin β3 was not detectable in the MDA-MB-231BO cells, and barely detectable in the MDA-MB-231P cells. It was expressed in detectable levels in MDA-MB-231BR cells and was slightly down regulated in reduced glucose. Error bars represent ± standard deviation. Note: β-Actin was used in all the blots as a protein loading control.</p
<p>(A) Cyclohexamide treatment for a period of 24 hrs depleted VEGFR2 protein from all thre... more <p>(A) Cyclohexamide treatment for a period of 24 hrs depleted VEGFR2 protein from all three cell lines. The blot in this panel demonstrates that cyclohexamide depleted VEGFR2 from the MDA-MB-231BO cells (regularly expressing the highest level of VEGFR2 among the three cell lines) whether they were hypoglycemic or hyperglycemic (lane 1 and 3). The control cyclohexamide negative samples –glu −CHX and +glu -CHX showed detectable VEGFR2 expression (lane 2 and 4). (B) VEGFR2 was completely depleted from the three cell lines when grown in hypoglycemia in the presence of CHX (lane 2). The samples in lanes 3 and 5 are cells exposed to hypoglycemia for 24 hrs without CHX and glucose was restored for 1 hr (lane 3) or 3h (lane 5), respectively. Comparing the former two lanes (3 and 5) shows clearly that the VEGFR2 band was increasing in molecular weight as the time of glucose exposure increased; this is very prominent in the middle (MDA-MB-231BO) and bottom blots (MDA-MB-231BR). The samples in lane 4 and 6 were glucose restored for 1 hr and 3 hrs respectively with CHX added at the time of glucose restoration. After 3 hr of CHX and glucose restoration of MDA-MB-231P cells, the VEGFR2 band was not detectable (lane 6- P), however the MDA-MB-231BO and MDA-MB-231BR cells (lane 6 BO, lane 6 BR) expressed a detectable VEGFR2 band. Note: β-Actin was used in all the blots as a protein loading control. (C) Stability of NRP-1 expression under low glucose and CHX treatment. NRP-1 was detected in the same samples as in the previous panel (B) and it did not show a profound decrease in its expression in both MDA-MB-231BO and MDA-MB-231BR. However in MDA-MB-231P samples the NRP-1 band pattern was changed after 24 hrs of hypoglycemia in the presence of CHX (lane 2). The NRP-1 band was also decreased in the glucose-restored samples with CHX (added at the time of restoration) (lanes 6, 8, and 10). (D) The blot shows that in MDA-MB-231BR cells the NRP-1 protein was resistant to CHX treatment for up to 72 hrs of exposure. Note: β-Actin was used in all the blots as a protein loading control.</p
<p>(A) Western blots show the difference in VEGFR2 and phospho-VEGFR2 in MDA-MB-231 parenta... more <p>(A) Western blots show the difference in VEGFR2 and phospho-VEGFR2 in MDA-MB-231 parental (P), bone metastatic (BO) and brain metastatic (BR) variants growing in DMEM containing 25 mM glucose. (B) Graph represents the mean of four independent ELISA experiments measuring the total VEGF-A secreted in the conditioned media, (error bars represent ± standard deviation). Statistical analysis showed no significant difference in the amount of VEGF-A secreted when the MDA-MB-231P and MDA-MB-231BO cells were grown under the different glucose concentrations. However, MDA-MB-231BR cells produced significantly higher levels of VEGF-A (p = 0.012) when grown in DMEM containing 25 mM of glucose compared to 0 mM of glucose. Figure abbreviations P: MDA-MB-231 Parental, BO: MDA-MB-231Bone and BR: MDA-MB-231Brain; cell lines were grown in DMEM 0, 5 and 25 mM glucose for 24 hrs without FBS. (C) VEGFR2 expression in MDA-MB-231P, MDA-MB-231BO and MDA-MB-231BR cells. The 147 kDa band was the dominant band expressed at 0 mM glucose. The regular double bands (200 kDa and 230 kDa) were detected in all the cells at 25 mM glucose. The cells expressed the bands differentially when placed in 5 mM glucose media. The MDA-MB-231P cells produced the 200 and 230 kDa forms; the MDA-MB-231BO cells produced the 147 and 200 kDa forms while the MDA-MB-231BR cells did not express any of the mature glycosylated bands at 5 mM glucose concentration. The bottom blot shows the expression of VEGFR1 was constant and did not change among the cell lines nor under the different glucose conditions. (D) Blot shows MDA-MB-231BR cells produced higher levels of GLUT-1 protein than the other cell lines. GRP78 had the same expression pattern among the different cells and its expression had an inverse relationship with glucose concentration. Note: β-Actin was used in all the blots as protein loading control.</p
<p> <b>hrs.</b> (A) The graph (left) shows real time PCR results for the positi... more <p> <b>hrs.</b> (A) The graph (left) shows real time PCR results for the positive expression of E-cadherin in MCF-7 and BT-474 cell lines; the grey and black bars represent two repeated independent experiments. However, MDA-MB-231P and its two variant cell lines did not express detectable E-cadherin message or protein (right); (B) Representative blots (left) and quantification (right) shows that MDA-MB-231P and MDA-MB-231BO cells expressed higher levels of N-cadherin compared with the MDA-MB-231BR cells, and that N-cadherin in the former cells was significantly reduced under hypoglycemia. γ, β and α-Catenins were all reduced under hypoglycemic conditions and their expression was significantly decreased in the two MDA-MB-231 variant cells only. Error bars represent ± standard deviation. Note: β-Actin was used in all the blots as a protein loading control.</p
Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism ... more Breast cancer (BC) arises commonly in women with metabolic dysfunction. The underlying mechanism by which glycemic load can exert its action on tumor metastasis is under investigated. In this study we showed that glycemic microenvironment alters the expression of three classes of proteins, VEGF and its receptors, cell to cell, and cell to extracellular matrix (ECM) adhesion proteins in MDA-MB-231 parental cells and its two metastatic variants to the bone and brain (MDA-MB-231BO and MDA-MB-231BR, respectively). Using western blotting, we showed that VEGFR2 levels were higher in these variant cells and persisted in the cells under extreme hypoglycemia. Hypoglycemia did not alter VEGFR2 expression per se but rather suppressed its posttranslational glycosylation. This was reversed rapidly upon the restoration of glucose, and cyclohexamide (CHX) treatment demonstrated that this deglycosylated VEGFR2 was not a product of de-novo protein synthesis. VEGFR2 co-receptor Neuropilin-1 was up-regulated four-fold in all MDA-MB-231 cells (parental and two variants) compared to VEGFR2 expression, and was also susceptible to glycemic changes but resistant to CHX treatment for up to 72 hrs. Hypoglycemia also resulted in a significant decrease in specific catenin, cadherin, and integrin proteins, as well as cellular proliferation and colony forming ability. However, MDA-MB-231BR cells showed a unique sensitivity to hypo/ hyperglycemia in terms of morphological changes, colony formation ability, integrin b3 expression and secreted VEGF levels. In conclusion, this study can be translated clinically to provide insight into breast cancer cell responses to glycemic levels relevant for our understanding of the interaction between diabetes and cancer.
2019 1st International Conference on Advances in Science, Engineering and Robotics Technology (ICASERT)
With the ever increasing population of the world, diseases and their possibilities are also incre... more With the ever increasing population of the world, diseases and their possibilities are also increasing at an alarming rate. As time passes by, diagnosing diseases and providing appropriate treatment at the right time has become quite a challenge. Heart diseases have been a major cause of death worldwide. Therefore, this research has been focused on finding an efficient way to predict the chances of a heart failure and accordingly, recommend appropriate medicines to aid cardiologists in quicker decision making. The research includes finding the correlations or associations between the various medical profiles of the patients by utilizing the standard techniques of exploratory analysis and hence using the attributes suitably to predict the chances of a heart failure, as well as the medicine recommendations. A comparative study has also been included which shows the various attained accuracy rates of different machine learning algorithms including - Logistic Regression, Naïve Bayes, Decision Tree, Linear Support Vector Classifier, Random Forest, and Gradient Boosting Classifier. The Apache Spark framework has been used in order to make the system capable of handling big data.
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