5 Latest Advancements in Cancer Research using Western Blotting and qPCR

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Azure Biosystems develops instrumentation to support a broad array of experimental approaches important to biomedical research.

Here we highlight five recent publications using Azure’s imaging and real-time PCR systems to advance cancer research.

1. How cancer cells evade ferroptosis

Liu et al investigated how cancer cells evade ferroptosis, a process of iron-dependent cell death that presents a promising anticancer therapeutic strategy (5). Cysteine depletion induces ferroptosis. The tumor environment is low in cysteine so cancer cells make cysteine by the reverse transsulfuration pathway which requires the enzyme cystathione b-synthase (CBS).

Chemiluminescent Western blots were imaged on the Azure c500.
Figure 4a from Liu et al, ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer showing changes in protein expression in colorectal cancer cells cultured in a cystine restricted medium for 24 hours. Chemiluminescent Western blots were imaged on the Azure c500. Licensed under CC BY 4.0.

The authors investigated how colorectal tumor cells regulate CBS. Western blots, imaged on the Azure c500 imaging system, were used to study changes in protein expression due to cysteine restriction and after knockdown of activating transcription factor 3 (ATF3). The study found ATF3 positively regulates CBS, suggesting that interfering with or blocking this signaling might enhance ferroptosis-based cancer therapy. Combined with an amino-acid restricted diet, the approach could present a new way to treat colorectal cancer.

Since the release of this publication, the c500 Imager used has been succeeded by the Azure 500 Imager, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this fluorescent imager today.

In addition to assessing miR-21 levels by qPCR, levels of PTEN and PDCD4 proteins were assessed by Western blots imaged on the Azure 600. The study found miR-21 expression to be increased in breast cancer but not correlated with age, ethnicity, or cancer subtype, suggesting it is a suitable biomarker across a variety of patient types.

2. Detecting changes in the expression of Bcl-2 in cells treated with novel nanocarriers containing anticancer agents

Tarvirdipour et al used the Sapphire Biomolecular Imager to image Western blots detecting changes in the expression of Bcl-2 in cells treated with novel nanocarriers containing anticancer agents (1). These authors developed self-assembling peptide nanocarriers designed to be taken up by cells and targeted to the nucleus while escaping endosomes without any additional modification.

Since the release of this publication, the Sapphire used has been succeeded by the Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins. Learn more about this groundbreaking new modular imager today.

The nanocarriers were loaded with a photosensitizer and an antisense RNA nucleotide against BCL-2. The Western blot experiments demonstrated that the combined photodynamic and gene therapies reduced Bcl-2 expression to a greater extent than either therapy alone. The combined therapy also resulted in higher cytotoxicity.

3. Response of the transcriptome of neuroblastoma cells to knockdown of the DDR2 gene

Vessella et al used bulk RNA sequencing to study the response of the transcriptome of neuroblastoma cells to knockdown of the DDR2 gene when the cells were grown on collagen-coated substrates (2). The authors also investigated the effects of increasing substrate stiffness. Collagen activates DDR2, a cell surface receptor that appears to be dysregulated in many cancer types. Increased extracellular matrix (ECM) stiffness has been reported to influence cancer metastasis and the authors hypothesized DDR plays a role in cancer cells mechanosensing of their environment.

Real-time PCR on the Cielo was used to confirm the RNA-seq results; reverse-transcription qPCR reactions were performed in 96-well plates with accumulated product detected using SYBR green. The analysis surprisingly found no transcriptome changes in response to ECM stiffness. The authors propose the ECM affects cancer cells via DDR2 signaling and mechanosensing of substrate stiffness via distinct mechanisms.

4. The effect of a combination therapy on castration-resistant prostate cancer

Fischetti et al studied the effect of a combination therapy on castration-resistant prostate cancer (CRPC) (3). CPRC has poor prognosis because the cancer is resistant to androgen receptor pathway inhibitors and to immunotherapy. The authors investigated the combination of enzalutamide, an androgen-deprivation therapy (ADT)/androgen receptor pathway inhibitor (ARPI), in combination with an inhibitor of EZH2, a repressor of transcription, with the aim of restoring hormone sensitivity and improving the antitumor immune response. The combination was tested in vitro in multiple cell lines and in vivo in a mouse model.

As part of the in vitro experiments, expression of androgen receptor, EZH2, and tri-methylation of histone H3 lysine 27 were confirmed by Western blots imaged on the Azure 600 imaging system. The study results suggest that the combination therapy presents a promising avenue for future clinical investigation and may allow new immunotherapy approaches in prostate cancer.

5. Whether breast cancer expression differs with ethnicity, age, or treatment status among Malaysian women

Wong et al report the expression of miR-21 in breast cancer cases among Malaysian women, investigating whether expression differs with ethnicity, age, or treatment status (4). miR-21 is an oncogenic miRNA that is frequently upregulated in breast cancer and whose expression is associated with more advanced disease. miR-21 is proposed to act via downregulation of tumor suppressor genes PTEN and PDCD4. The majority of studies of miR-21 have involved homogeneous populations, making conclusions about relative expression levels between populations difficult. The authors aimed to study expression of the gene in the multi-ethnic Malaysian population whose access to and quality of care should be comparable.

In addition to assessing miR-21 levels by qPCR, levels of PTEN and PDCD4 proteins were assessed by Western blots imaged on the Azure 600. The study found miR-21 expression to be increased in breast cancer but not correlated with age, ethnicity, or cancer subtype, suggesting it is a suitable biomarker across a variety of patient types.

Ready to learn more about how easy Western blotting is by using an Azure Imager?

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Two scientists looking at multiplex fluorescent Western blot on Azure 600 Western blot imager
Revolutionizing the way you Western blot! Azure Imagers are high performance Western blot imaging systems capable of NIR fluorescence, visible fluorescence, and chemiluminescence.
SOURCE
  1. Tarvirdipour S, Skowicki M, Maffeis V, Abdollahi SN, Schoenenberger C, Palivan CG. Peptide nanocarriers co-delivering an antisense oligonucleotide and photosensitizer elicit synergistic cytotoxicity. J Colloid Interface Sci. 2024;664:338-348.
  2. Vessella T, Xiang S, Xiao C, et al. DDR2 signaling and mechanosensing orchestrate neuroblastoma cell fate through different transcriptome mechanisms. FEBS Open Bio. doi: 10.1002/2211-5463.13798. Published online ahead of print.
  3. Fischetti I, Botti L, Sulsenti R, et al. Combined therapy targeting AR and EZH2 curbs castration-resistant prostate cancer enhancing anti-tumor T-cell response. 2024. doi: 10.2217/epi-2023-0374. Published online ahead of print.
  4. Wong SR, Pei CP, Islahuddin MTM, et al. miR-21 Expression and its correlation with demographics, subtypes, and tumour suppressor genes; PTEN and PDCD4 in breast cancer tissues in Malaysia. 2024;8(1):59-73.
  5. Liu J, Lu X, Zeng S, et al. ATF3-CBS signaling axis coordinates ferroptosis and tumorigenesis in colorectal cancer. Redox Biol. 2024;71:103118.

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