Research ArticleExperimental Studies

Effects of Hypoxia on Proliferation and Apoptosis of Osteosarcoma Cells

SHANG-YOU YANG, ELKA GARCIA, WILL XIA and ANYU WANG
Anticancer Research October 2021, 41 (10) 4781-4787; DOI: https://doi.org/10.21873/anticanres.15293

Abstract

Background: Hypoxia can happen during solid tumor growth including osteosarcoma. This study investigated the relationship of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) on osteosarcoma cell growth and apoptosis under hypoxic conditions. Materials and Methods: Human osteosarcoma cells were cultured under normal or hypoxic conditions. Inhibitors of HIF-1α and VEGF were applied to the cells separately or in combination to block the respective proteins. Cell proliferation and apoptosis were examined by MTT and TUNEL assays, and real-time PCR and ELISA were performed for mRNA and protein expression. Results: There was a dramatic decrease of cell proliferation and an elevation of apoptosis under hypoxia. Blockage of HIF-1α and VEGFR enhanced the cell growth retardation and promoted apoptotic changes. Moreover, blockage of HIF-1α significantly eliminated the expression of VEGF in the cell culture media, and vice versa. Conclusion: HIF-1α and VEGF work closely in regulating osteosarcoma cell growth under hypoxic conditions and blockage of either of them may subsequently influence the presence of the other.

Key Words:

Osteosarcoma is one of most common primary bone malignancies in children and young adults. In the past several decades, the application of adjuvant chemotherapy and wide surgical resection has improved the osteosarcoma patients’ initial treatment outcomes. However, the 5-year survival rate remains around 65%, while it is as low as 30% or less after recurrence or metastasis occurs (1). Osteosarcoma often migrates to other parts of body, especially to the lungs in 6 months to a year without formal treatment (2). Therefore, more effective therapies and novel therapeutic targets are critically needed to improve survival rates. Treatment resistance appears to be a main reason of failure during current osteosarcoma therapies.

Hypoxia is a common phenomenon in growing solid human tumors including osteosarcoma. Thomlinson first reported in 1955 that many malignant tumor tissues possessed hypoxic areas (3). Later studies have suggested that many tumors including osteosarcoma grow so quickly that often exceed their own vascular supply to sequentially lead local low oxygen tension (hypoxia) and nutritional deficiency (4). Those tumor cells under low oxygen tensions express growth factors and mediators to promote neovascularization, and further result in accelerated tumor proliferation and metastasis (4). In addition, studies in animal models using transcutaneous CO2 therapy or nanoparticles for reoxygenation of experimental solid tumors, including osteosarcoma, obtained outcomes of decreased tumor growth and metastatic potential (5, 6). Many important transcription factors and mediators have been identified in the hypoxic micro-environment, and the expression of these hypoxia markers correlates with poor patient outcomes and treatment resistance in osteosarcoma (7, 8). One of the most important hypoxia-associated factors is hypoxia-inducible factor-1 (HIF-1) (9). HIF-1 activates a series of genes including vascular endothelial growth factor (VEGF) that directly contribute to tumor aggressiveness. The hypothesis of this investigation is that low oxygen tension during osteosarcoma progress would promote HIF-1 expression and coupled VEGF expression, which would be critical in tumor cell survival and growth. In the current study, osteosarcoma cells were cultured to simulate the hypoxic condition to examine the variations of HIF-1 and VEGF expressions on tumor cell proliferation and apoptosis in this in vitro osteosarcoma model (10).

Materials and Methods

Maintenance of osteosarcoma cell culture. A human osteosarcoma cell line (CRL-1547, ATCC, Gaithersburg, MD, USA) was cultured at 37°C in 5% CO2, in Eagle’s MEM medium with 10% bovine fetal serum (FBS), 2 mM L-glutamine and 1.5 g/l sodium bicarbonate, 0.1 mM non-essential amino acids, 1.0 mM sodium pyruvate, 100 U/ml penicillin and 100 μg/mL streptomycin (all reagents from ThermoFisher Scientific, Waltham, MA, USA). The cell culture was maintained with medium changes at 3-day intervals till 90% confluence. Subculture of the cells was performed by 0.25% trypsin and 0.03% EDTA for cell dissociation followed by centrifugation (200 × g for 10 min). Cell viability and quantification were checked by staining a fraction of the cell pellet with 0.5% (w/v) trypan blue under a light microscope. Cells were cultured either in normoxic conditions (20% O2 concentration), or under hypoxia (GasPak EZ Gas Generating Sachet, BD Life Sciences, Franklin Lakes, NJ, USA) for 72 h. Oxygen levels in the hypoxia group were maintained at 3%. For blockage studies, 40 μM of KC7F2 (HIF-1α inhibitor; 4324/10, R&D Systems, Minneapolis, MN, USA) and 10 μM of SU4312 (VEGFR inhibitor; 58567, Sigma-Aldrich, St. Louis, MO, USA) were added to the cell cultures, respectively or in combination.

Proliferation of osteosarcoma cells. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was performed to determine the tumor cell proliferation under hypoxic or normoxic conditions. Osteosarcoma cells at 1×105 cells/well were exposed to HIF-1α and/or VEGF inhibitors under different oxygen tensions for 3 days. Fresh medium containing MTT at final concentration of 0.5 mg/ml was then applied to culture for a 4-h incubation at 37°C. The enzyme reaction was stopped by 10% SDS at end of incubation and optical density (OD) readings were obtained at 590 nm using a plate spectrophotometer (11). TUNEL assay for apoptosis. Terminal DNA Breakpoints in situ 3-Hydroxyl End Labeling (TUNEL) assay was adopted for assessment of cell apoptosis (Roche In Situ Cell Death Detection kit, Sigma-Aldrich Chemicals, St. Louis, MO USA). Briefly, ATCC-1547 cells at 2×104 cells/well were cultured on an 8-well chamber slide overnight at 37°C prior to being treated with HIF-1α and/or VEGF inhibitors under different oxygen concentrations for 3 days. Prior to apoptotic cell staining, cells were fixed in a freshly prepared 4% paraformaldehyde solution for 60 min, followed by rinses with PBS. After applying a permeabilization solution for 2 min on ice and a PBS wash, cells on the slide were covered with 50 ul of TUNEL reaction mixture per well for incubation of 60 min at 37°C in a humidified chamber in the dark. A slide was then applied with 50 ul/well of converter-AP for 30 min at 37°C in a humidified chamber. After rinsing, 100 μl of substrate solution was added for a 10-munite incubation in the dark at room temperature (RT). Slides were then mounted with glass coverslips and analyzed under a microscope. Quantification of the apoptotic cells was expressed as apoptotic index, which represents the ratio of the TUNEL-positive cells over total cells (12).

Real-time RT-PCR analysis. As detailed previously (10), mRNA expression profiles of certain genes of osteosarcoma cells at different oxygen concentrations were quantified using real time reverse-transcription polymerase chain reaction (RT-PCR). Following RNA isolation, cDNA was obtained by reverse transcription from 0.5 μg of total RNA in a DNA Thermal Cycler (Veriti 96 well Thermal Cycler, AB Applied Biosystems, Bedford, MA, USA). Primers were designed using Primer 3 (http://bioinfo.ut.ee/primer3-0.4.0/). The following primers were manufactured from ThermoFisher Scientific: HIF-1α forward 5’-actagccgaggaagaactatgaa-3’, HIF-1α reverse 5’-tacccacact gaggttggtta-3’; and a pair of VEGF primers: forward 5’-cccactgagga gtccaacat-3’, reverse 5’-tttcttgcgctttcgttttt-3’. The housekeeping gene 18S was used as internal control. Real-time gene expression levels of HIE-1α, VEGF and 18S on cDNA samples were dynamically recorded using the Step One Plus Real-Time PCR System (AB Applied Biosystems).

Cytokine quantification by ELISA. Human/Mouse Total HIF-1α ELISA Kit (R&D, DYC1935-2) and the Human VEGF Mini ELISA Development Kit (PeproTech US, Cranbury, NJ, USA 1012010-M) were adopted to quantify the protein levels of HIF-1α and VEGF in the conditioned media of the osteosarcoma cells after treatments. The ELISA plate was prepared by overnight coating with capture antibody on each well of a 96-flat-bottom-well plate at 4°C. The collected condition media of 100 μl each was add to the air-dried plate in triplicates per group. Known concentrated HIF-1α or VEGF were also sequentially diluted into wells of the plate as protein standards. The plate was incubated overnight at 4°C. After washing, the detection antibody was applied at 2 μg/ml to each well for 2 h at room temperature. Following streptavidin-HRP tagging and washing steps, color development was observed by addition of 100 μl of 1×TBM solution at room temperature in the dark for 5 min. After addition of 50 μl of 1N hydrochloric acid to stop the development, the plate was read at 450 nm on a micro-plate reader (SpectraMax+ 384, Molecular Devices, Sunnyvale, CA, USA), The estimated VEGF or HIF-1α levels were calculated against standard curves. Each experiment was repeated three times.

Statistical analysis. Data among testing groups were expressed as means±standard error of the mean (SEM). Independent t-test and one-way ANOVA were utilized for statistical analysis (IBM SPSS, Chicago, IL, USA, Ver. 23). In all statistical evaluations, p<0.05 was considered to be statistically significant.

Results

Cell proliferation patterns in hypoxic environment. After osteosarcoma cells were cultured for 72 h, cell proliferation was assessed by MTT assay. A significantly slower proliferation pattern was observed in osteosarcoma under hypoxic conditions compared to those in normoxia (Figure 1A, p<0.05). In addition, selectively inhibiting the functions of VEGF and/or HIF-1 by chemical inhibitors significantly decreased cell proliferation in normoxic conditions (Figure 1B, p<0.05). Since all groups in hypoxic conditions already exhibited retarded cell proliferation, further inhibition treatment only resulted in an additional diminished cell growth in the VEGFR inhibition group compared to controls (Figure 1B, p<0.05).

Figure 1.
Figure 1.

MTT assay for cell proliferation. (A) Osteosarcoma cells under normoxia and hypoxia for 72 h. (B) Cells were treated with KC7F2 (HIF-1α inhibitor) and SU4312 (VEGF inhibitor), respectively or in combination. *p<0.05.

Apoptotic changes under hypoxic conditions. Under normal oxygen conditions, osteosarcoma cells exhibited dramatically increased apoptotic changes after VEGFR and/or HIF-1 inhibition treatments, in comparison with the non-treated controls (Figure 2A-D). In hypoxic culturing conditions, total cell counts from all tested groups were markedly decreased compared to cells in normoxia (Figure 2E); although it did appear that blockade of VEGF/HIF-1 induced further cell death (Figure 2F-H). Indeed, only minimal cells survived after combination treatment of VEGFR and HIF-1 inhibitors under hypoxic conditions (Figure 2H). Figure 3 summarizes the computerized image analysis of the TUNEL-positive staining, where hypoxic conditions significantly increased apoptotic cells compared to cells in normoxia (p<0.05). In addition, blockage of HIF-1α, VEGFR, or a combination of both resulted in a significant increase in apoptotic cells compared to controls (Figure 3); there were essentially no cells in the culturing wells under hypoxic conditions with the added treatment of both inhibitors.

Figure 2.
Figure 2.

TUNEL assay for apoptotic cell identification. Top panel shows osteosarcoma cells under normoxic conditions; lower panel shows cells in hypoxic conditions. A and E: non-treated control; B and F: treated with KC7F2; C and G: treated with SU4312; D and H: treated with KC7F2 and SU4312.

Figure 3.
Figure 3.

Quantification of apoptotic cells after HIF-1α and/or VEGF blockage in normal and hypoxic culture conditions. *p<0.05 vs. non-treated controls.

HIF-1α and VEGF gene expression profiles in different oxygen concentrations. Gene expressions of HIF-1α and VEGF had a significant difference under hypoxic conditions. Under normoxic condition, expression of HIF-1α could be diminished by application of HIF-1α and VEGF inhibitors. Significantly over-expressed HIF-1α mRNA was noticed in cells after culture in hypoxia compared to those in normoxia (Figure 4A). This over-expression of HIF-1α could not be corrected by addition of the HIF-1α inhibitor KC7F2 to the cultures. However, its expression was effectively inhibited by the treatment of VEGFR inhibitor SU4312 (p<0.05, Figure 4A). Similarly, combination of KC7F2 and SU4312 to the cultures significantly suppressed HIF-1 expression. Interestingly, both inhibitors (KC7F2 and SU4312) effectively inhibited mRNA expression of VEGF in various oxygen conditions (p<0.05, Figure 4B).

Figure 4.
Figure 4.

Real-time PCR to quantify the expression changes of Hif-1α (A) and VEGF (B) at normoxic or hypoxic conditions.

The protein levels of HIF-1α and VEGF under hypoxia. The data suggested an elevated production tendency in HIF-1α protein levels in collected culture media under hypoxia compared to normoxia, although statistical significance was not reached (Figure 5A). However, the VEGF protein levels appeared significantly decreased in the culture medium of the tumor cells in hypoxia (Figure 5C, p<0.05). Both HIF-1α and VEGFR inhibitors significantly suppressed HIF-1α protein expression at normal or low oxygen concentrations, although more dramatic inhibitory effects were noticed under hypoxic conditions (Figure 5B). Addition of the chemical inhibitors against VEGFR or/and HIF-1α significantly further diminished the VEGF levels in the culture media at either normoxic or hypoxic conditions (Figure 5D).

Figure 5.
Figure 5.

Protein levels of HIF-1α and VEGF were quantified among samples using ELISA. *p<0.05 vs. controls.

Discussion

Sustained tumor cell proliferation appears to be one of the hallmarks of cancer. Supressing cell proliferation and/or inducing cancer cell apoptosis is a crucial step in cancer treatment. Osteosarcoma has been the most common primary bone malignancy. Essentially all solid tumors depend on vascular networks for oxygen and nutrition to support their growth. In this regard, a hypoxic environment exists during the tumor fast growth stage. Hypoxia, which is the result of an imbalance between tumor cell proliferation and blood supply, is one of the most important pathological characteristics of solid tumors (13, 14). Hypoxia markers such as hypoxia-inducible factor-1 alpha (HIF-1α) and vascular endothelial growth factor (VEGF) can be detected in osteosarcomas (7, 15, 16) and the presence of these markers correlates with poor treatment outcomes, suggesting that hypoxia has an important role in osteosarcoma (8, 17).

Growing evidence have demonstrated that intra-tumoral low oxygen tension can promote invasive tumor growth and distant metastasis, although debate exists on the influence of hypoxia in promoting tumor cell apoptosis or rather inhibiting cells from apoptosis. In the current study, a GasPak EZ Gas Generating Sachet was adopted to simulate a hypoxic microenvironment for human osteosarcoma cells. It is apparent that the cell proliferation was repressed, and ubiquitous TUNEL-positive apoptotic cells were observed under the hypoxic microenvironment. Recent clinical and experimental investigations have suggested that intra-tumor hypoxia may play a key role in the process of solid tumor invasive growth and metastasis (18). The increased malignancy of hypoxic tumors has been attributed to the altered cellular metabolism and their resistance to chemotherapy or radiotherapy (19, 20). Indeed, cancer cells must compensate under low oxygen tension condition to survive. The adaptive responses of tumor cells to hypoxia eventually increase their capacity for angiogenesis, invasion and metastasis.

HIF-1α is a transcription factor induced by hypoxia that regulates gene expression in response to hypoxic conditions (21). Experiments show that the transcript activity of HIF-1α increases under the hypoxic microenvironment that in turn, regulates the expression of downstream genes (9, 22). However, it is still unknown if HIF-1α can promote tumor cell apoptosis or anti-apoptosis due to controversial research results. Some studies have indicated that overexpression of HIF-1α may promote apoptosis by activating Bcl-2 and Bcl-Xl, or maintaining the stability of p53 (23). Contrarily, another study reported that HIF-1α could up-regulate VEGF and GLUT1 expression to endure tumor cells resistant to apoptosis (24). Investigators have also studied other microenvironmental factors such as acidic pH and interstitial fluid pressure to see if hypoxia can have additive effects on the survival and invasiveness of osteosarcoma cells. Matsubara et al. reported that HIF-1α was significantly overexpressed from cells cultured at pH 6.8 compared to those at pH 7.4, but the collective effects of these physicochemical changes were inconclusive on different osteosarcoma cell lines (25).

The current investigation showed a significant elevation of HIF-1α gene expression under hypoxia, which is consistent with the report of Cheng et al. (9). KC7F2, a compound known as an inhibitor of HIF-1α, is thought to act via down-regulation of HIF-1α protein synthesis, but not influence HIF-1α gene expression. We adopted this compound to block the function of HIF-1α in the osteosarcoma cell cultures. In normoxia, cell proliferation decreased when HIF-1α protein level was suppressed by KC7F2. However, cell proliferation was not influenced by the fluctuation of HIF-1α under hypoxia, which suggested that HIF-1α failed to further affect osteosarcoma cell proliferation in a low oxygen concentration. However, inhibition of HIF-1α did result in increased cell apoptosis, both under hypoxia and normoxia. This finding appears in agreement with Dai et al. (24), suggesting that HIF-1α is a critical regulator in the survival of osteosarcoma cells.

VEGF is a major mediator for angiogenesis and has been considered crucial in carcinogenesis and remote metastasis (26). Ma et al. reported that recombinant human relaxin-2 effectively promoted the proliferation, invasion and angiogenesis of osteosarcoma cells by up-regulating pAKT-dependent VEGF expression (27). In the current study, we found that there was a decreased VEGF gene expression under hypoxia that also coincided with a suppressed cell proliferation. Indeed, by blocking VEGF receptor with its chemical inhibitor SU4312, a significant downregulation of VEGF gene expression was noticed in both normoxic and hypoxic microenvironments, which correlated with the suppressed tumor cell proliferation. We deduced that VEGF can directly promote cell proliferation, down regulating its expression would result in halting cell proliferation. Further, blockage of VEGFR resulted in elevated apoptotic cells, and combination of both blockage of HIF-1α and VEGFR indicated synergistic effects in promoting cell apoptosis.

Overall, this study suggests that cell proliferation and apoptosis occur concurrently during tumor development in this type of osteosarcoma cells. HIF-1α may significantly induce an anti-apoptotic effect and VEGF promotes cell proliferation. Blockade of both HIF-1α and VEGF may be a potential therapy for osteosarcoma.

Acknowledgements

The Authors wish to thank Ms. Zheng Song for her excellent technical assistance. The majority of the data presented here are also included in one of the student authors – Elka Garcia’s Master Thesis as part of a requirement for the Wichita State University Graduate School. This work was supported by research grants from Kansas Flossie E. West Memorial Foundation and Wichita Research & Medical Education Foundation.

Footnotes

  • ↵Authors’ Contributions

    AW and EG performed the experiments and wrote the manuscript. SYY and WX conceived the study. AW and SYY analyzed data. SYY participated in manuscript preparation and critical editing.

  • Conflicts of Interest

    The Authors declare no conflicts of interest.

  • Received April 2, 2021.
  • Revision received August 25, 2021.
  • Accepted August 31, 2021.

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Effects of Hypoxia on Proliferation and Apoptosis of Osteosarcoma Cells
SHANG-YOU YANG, ELKA GARCIA, WILL XIA, ANYU WANG
Anticancer Research Oct 2021, 41 (10) 4781-4787; DOI: 10.21873/anticanres.15293
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