e-book Glycolysis: Review Notes in Biochemistry (Quick Review Notes)

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Methods: OC cell lines with Pim1 overexpression or knockdown were constructed with lentivirus transduction. Cell Counting Kit-8, colony formation, glycolysis stress test and in vivo mice models were carried out to assess the effect of Pim1 on OC biological functions. Co-immunoprecipitation assay coupled with western blot were performed to explore the intrinsic mechanisms of Pim1 in OC. Bioinformatic analysis was then performed to evaluate the expression and prognostic value of Pim1.

Results: We present the first evidence that silencing or overexpressing Pim1 can suppress or promote, respectively, OC cell proliferation. Furthermore, we demonstrated that Pim1 can significantly enhance glycolysis in OC cells. In vivo experiments further confirmed that knockdown of Pim1 inhibits the growth of tumors derived from the SKOV3 cell line. To search for the underlying molecular mechanism, we examined the effect of Pim1 on MYC, a pivotal gene in glycolysis, and observed that Pim1-mediated phosphorylation of c-Myc activated the expression of glycolysis-associated key genes such as PGK1 and LDHA.

Moreover, we found that the Pim1 inhibitor SMI4a induced chemosensitization to cisplatin.

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Clinically, Pim1 was also overexpressed in OC and correlated with poor overall survival by bioinformatics analysis. Conclusion: Together, these results suggest that Pim1 contributes to proliferation and glycolysis in OC via interaction with MYC and may serve as a potential target in the treatment of OC patients. Ovarian cancer OC is the fifth leading cause of cancer-related death in women worldwide. Aerobic glycolysis, which is also known as the Warburg effect and is a hallmark of cancer, is characterized by the increased conversion of glucose into lactate irrespective of oxygen availability and is an important process that provides numerous intermediates for cell survival and new biomass building in cancer.

Thus, a better understanding of the underlying mechanisms of glycolysis in OC might aid in the discovery of novel treatment opportunities that are urgently needed. Previous evidence has shown that overexpression of Pim1 in various human cancers such as breast cancer and glioblastoma is well correlated with processes of cancer progression, including cell proliferation, cell cycle arrest, apoptosis, migration, invasion, and drug resistance.

Glycolysis

However, the role and underlying mechanisms of Pim1 in growth, development, and aerobic glycolysis in OC remain unclear. This study was undertaken to test the hypothesis that Pim1 provides a proliferative advantage and regulates glycolysis in OC. Furthermore, we examined the impact of Pim1 on c-Myc to explore the underlying molecular mechanism. The control vector pcDNA3. To generate stable cell lines, the pLKO.

Lentiviral particles were produced by cotransfection of the pLKO. G into HEKT cells in a ratio of A and cells were transfected with pcDNA3. All experiments were performed in triplicate. Cell proliferation curves were plotted using the absorbance at each time point. Cells were digested with trypsin into single-cell suspensions at 48 hours after transfection. After one week, the cells were fixed and stained with 0.

Triplicate wells were measured for each treatment group. Quantitative real-time PCR was performed as described previously.


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ACTIN was used as an internal control. The isolated proteins were probed with the indicated primary antibodies followed by incubation with HRP-linked secondary antibodies and detection using an ECL system Thermo Fisher Scientific. The animals were sacrificed after three weeks and the tumor bulks were harvested.

Step 2: Phosphoglucose Isomerase

The tumor specimens were preserved in liquid nitrogen. Immunohistochemical IHC analysis of Ki —1-AP; Proteintech was performed on 5 mm sections of paraffin-embedded tumors as previously described. Protein expression analyses of the indicated proteins were performed by western blot analysis. The data are presented as the mean with error bars indicating the SD. All statistical analyses were performed using SPSS Recent evidence shows that Pim1 can influence cancer cell proliferation in many different tumors. A and were selected for overexpression experiments due to their low endogenous expression of Pim1.

Then, we established stable cell lines with Pim1 overexpression and knockdown, and the efficiencies of overexpression and downregulation were validated by western blot analysis and qRT-PCR, respectively Figure 1B. Then, we utilized CCK-8 and colony formation assays to elucidate the potential effect of Pim1 on OC tumor cell proliferation. Figure 1 Pim1 promotes the growth of OC cells in vitro. Notes: A Pim1 protein expression, as assessed by western blot analysis in a panel of OC cell lines. D A colony formation assay was utilized to assess the effect of Pim1 on the colony formation ability of OC cells.

Previous literature reported that the proliferation of tumor cells is usually accompanied by metabolic changes.

Glycolysis- Lecture-1 | Biochemistry for Medics – Lecture Notes

Reprogrammed energy metabolism is considered a hallmark of cancer, and dysregulated energy metabolism in tumor cells is mainly attributed to mitochondrial dysfunction. Here, extracellular acidification rate ECAR and oxygen consumption rate OCR measurements were performed to investigate the cellular metabolic status. The OCR value reflects glucose mitochondrial oxidation. Moreover, the lactate colorimetric assay was adopted to assess cellular lactate production.

These results indicated the role of Pim1 in promoting glycolysis. Figure 2 Pim1 influences glucose metabolism and mitochondrial respiration in OC cells. E , F As expected, Pim1 can boost lactate production. All data are from a single experiment with three wells. Based on the abovementioned observations, we next investigated the effect of Pim1 on tumorigenicity in vivo by comparing tumor growth using a stable Pim1 knockdown cell line.

Knockdown of Pim1 significantly reduced the tumor growth in vivo in nude mice injected subcutaneously with tumor cells of the SKOV3-shPim1 cell line Figure 3A. In addition, the xenografts derived from Pim1-knockdown SKOV3 cells were smaller than those derived from control cells in both size and weight Figure 3B and C. In order to demonstrate an effect of Pim1 on the growth in vivo, we also conducted IHC of Ki, which is a proliferation index.

Results showed that Ki was lower in the Pim1 knockdown groups than in the control groups Figure 3D.

Glycolysis

Figure 3 Tumorigenicity of Pim1 in vivo. B The volumes of the growing tumors were measured every five days. Then, mice were killed 20 days after subcutaneous injection. C Bar graph analysis of the mouse tumor weights. Pim1-mediated phosphorylation of c-Myc activates the expression of glycolysis-associated key genes. Previous studies demonstrated that c-Myc is primarily responsible for metabolic reprogramming. We next investigated whether c-Myc participates in Pim1-mediated glycolysis in OC cells.

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Immunoblotting and co-immunoprecipitation assays were performed, and the results indicated that Pim1 interacts with c-Myc and phosphorylates c-Myc at S62 Figure 4A. Phosphorylation of c-Myc at the Ser62 position is thought to activate key enzymes in the glycolytic pathway. The expression levels of metabolic-related enzymes that did not change are shown in Table S3.

Moreover, the protein levels of these enzymes were assessed by western blot analysis.

This difference may be due to the existence of other mechanisms of Pim1 in regulating glycolytic pathways. Figure 4 Pim1-mediated phosphorylation of c-Myc activates the expression of glycolysis-associated key genes. Lysates were also subjected to co-immunoprecipitation co-IP with an IgG control. D Moreover, the protein levels of the associated enzymes were assessed when Pim1 was downregulated by siRNAs. Protein bands were also quantified by ImageJ software. These findings demonstrated that SMI4a may serve as a promising chemosensitizing target.

Abbreviation: OC, ovarian cancer.


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  • To determine the clinical relevance of Pim1 expression, we first analyzed Pim1 protein expression in clinical specimens from the Human Protein Atlas. Taken together, these results clearly suggest that Pim1 is aberrantly overexpressed in human OC tissues and is positively correlated with human OC progression.

    Figure 6 Pim1 is upregulated in human ovarian carcinoma specimens. Notes: A Pim1 expression in normal ovarian tissue and OC specimens.