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Gene & Protein in Disease Verteporfin therapy for triple-negative breast cancer

globally reported breast cancer cases. In comparison to 159 cells were maintained in DMEM with F12 at 37°C in a
the non-TNBC cohort, higher heterogeneity, increased 5% humidified CO incubator. All cells were supplemented
2
invasiveness, lower relapse-free and disease-free survival, with 10% (vol/vol) fetal bovine serum and 1% (vol/vol)
and increased cellular aggression are the basic features of antibiotic-antimycotic solution and routinely checked to
TNBC patients. The current therapeutic regime applied be free of mycoplasma contamination.
to TNBC patients involves combinatorial chemotherapy,
radiation therapy, and surgery, although patients with 2.2. Reagents and antibodies
similar clinicopathological variables exhibit differential Antibodies against β-ACTIN, YAP, TEAD, LATS1, p21,
outcomes [3,4] . p27, and caspase 3 were purchased from Santa Cruz
The hippo signaling cascade is a family of signal- Biotechnology (USA), antibodies against TAZ, CTGF,
responsive transcriptional co-activators highly conserved CYR61, p-YAP (Ser127), MYC, Cyclin D1, poly (ADP-
through the evolution wherein the components transduce ribose) polymerase (PARP), and cleaved caspase 3 were
[5]
signals to harmonize normal cellular architecture . purchased from Cell Signaling Technology (Danvers,
MA), antibodies against Ki-67 were purchased from
The upstream regulatory module of the Hippo pathway Abcam (UK). Anti-mouse, anti-rabbit secondary
governs the co-transcriptional activity of the transducers
YAP/TAZ in a negative mode . High expression of yes- antibodies, and chemiluminescence detection luminol
[6]
™
associated protein (YAP) is found to associate with poor reagents (SuperSignal West Pico Plus Chemiluminescent
prognosis and survival in liver cancer, non-small lung Substrate) were obtained from ThermoFisher Scientific
(Waltham, MA, USA). Alexa Flour 488 conjugated anti-
cancer, colorectal cancer, gastric cancer, melanoma, head- mouse secondary antibodies were purchased from Cell
and-neck cancer, squamous cell carcinoma, gliomas, and Signaling Technology (Danvers, MA). Benzoporphyrin
breast cancer [7-10] . While some studies point out the role of derivate VP was purchased from Sigma Aldrich, USA.
YAP as a tumor suppressor in breast cancer [11,12] , some have Small interfering RNA (siRNA) targeting YAP (siYAP) and
reported amplified expression of YAP in TNBC besides its control siRNA (siCN) were purchased from Santa Cruz
relation to poor survival [13,14] .
Biotechnology.
Verteporfin (VP) was approved for photodynamic
therapy in macular degeneration by the US Food and 2.3. Drug treatment and siRNA transfection
Drug Administration in 2000 . As a second-generation For VP treatment, cells at 70 – 80% confluence were used
[15]
benzoporphyrin derivative (BPD), it exists as an equal and incubated 24 h prior to conducting all functional
mixture of two regioisomers (BPD monoacid ring A) . assays. Cells at 60% confluence were taken for the siRNA
[16]
Upon activation with 680 – 700 nm wavelength intensity transfection according to the instructions provided by
light, VP produces reactive oxygen radicals and singlet the manufacturer. Cells were subjected to all further
oxygen, which act as a cytotoxic agent [15,16] . This idea experiments after 24 h of transfection.
of photosensitizer was then employed in animal tumor
models for effective vascular damage and tumor 2.4. Western blot assay
regression . However, a game-changing discovery by For the western blot assay, whole cell lysate was prepared
[17]
Liu-Chittenden et al. revealed the ability of VP to inhibit in RIPA lysis buffer and 1% protease inhibitor and
the co-transcriptional activity of the Hippo transducers in phosphatase inhibitor supplemented. The total protein
the absence of phototherapy, repurposing the usage of the was quantified using a BCA reagent kit (ThermoFisher
[18]
drug . Scientific, USA). 50 – 80 µg of proteins were used; equal
In the current study, our target is to investigate the concentrations of protein loading were ensured at each time
effect of VP on TNBC cells, specifically analyzing its of immunoblotting. Polyvinylidene fluoride membrane
impact on the unique phenotypical behavior of these cells. (Bio-Rad, USA) was used for the protein transferring.
We evaluated the anti-tumorigenic properties of VP on Blots were probed with corresponding antibodies against
the TNBC cells and elucidated the molecular mechanism the protein of interest, and β-actin was used as the internal
underlying its effects. control. Horseradish peroxidase-conjugated secondary
antibodies were used, and the chemiluminescent signal was
2. Materials and methods detected using the enhanced chemiluminescence reagent.
2.1. Cell culture and maintenance 2.5. Quantitative real-time PCR

MDA-MB-231, MCF7, and T47D cells were maintained in In order to perform quantitative real-time PCR, the total
Dulbecco’s modified Eagle’s medium (DMEM), and SUM- RNA from cells was extracted using Trizol (Ambion)

Volume 2 Issue 2 (2023) 2 https://doi.org/10.36922/gpd.0658