TGF- β downregulation overcomes gemcitabine resistance in oral squamous cell carcinoma

2.1 Cell lines and reagents

TCA8113 and Tb cells were maintained in DMEM containing 10% FBS at 37 ∘ C in a humid environment with 5% CO 2 .

Antibodies targeting phospho-Akt (Ser473), phospho-p65 (Ser536), and PARP were purchased from Cell Signaling (MA, USA). BCA Protein Assay was bought from Thermo (USA). All other reagents were bought from Sigma (USA).

2.2 The construction of retroviral vectors

The two micrograms of the resulting lentivirus transfer plasmids pCDH-shTGF- β 1/ β 2 and pCDH together with 1.5 mg psPAX2 vector and 0.5 mg pMD2.G vector, respectively, were co-transfected into HEK-293T cells (Invitrogen) in a 10 cm plate and incubated for 72 hours at 37 ∘ C and 5% CO2. Then, culture supernatants were centrifuged in Amicon ® Ultra-15 Centrifugal Filter Devices (3000 rpm, 30 minutes) for virus particle concentration.

TCA8113 and Tb cells were incubated in FBS-free medium as above for 48 hours. DMEM supplemented with 10 μ g/ml polybrene (Sigma) was refreshed daily. Next, 100 μ l of concentrated pCDH-shTGF- β 1/ β 2 lentivirus was mixed with 1ml of the culture medium.

2.3 Cell viability assays

TCA8113 and Tb cells cultured in 96-well plates were administered in various doses of gemcitabine (0, 2.5, 5, 10, 15, and 20 μ M) for 24 hours and 48 hours, respectively. Cell viability after treatment of TGF- β was also evaluated. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) assay was employed to assess cell viability and expressed relatively to control the values.

2.4 Western blot

TCA8113 and Tb cells cultured in 6-well plates were administered with gemcitabine (IC50) for 24 hours and 48 hours, respectively and lysed. Protein amounts in the lysates were assessed with the BCA Protein Assay Kit. Anti-phospho-Akt (p-Akt), anti-p-p65), anti-PARP and anti-GAPDH primary antibodies were used for blotting.

2.5 Real-time RT-PCR

Cell lysis was performed with TRIzol reagent and total RNA was obtained via chloroform extraction. Real-time RT-PCR was carried out with the Power SYBR Green RNA-to-CT 1-Step Kit. Sense and antisense primers were synthesized based on a report for human TGF- β 1, TGF- β 2, and β -actin mRNAs [17].

2.6 The measurement of the TGF- β 1 and TGF- β 2 levels by ELISA

TCa8113 and Tb cells cultured in 6-well plates were administered with gemcitabine (IC50) for 24 hours and 48 hours, respectively. Supernatants were obtained after 48 hours. Secreted TGF- β 1/ β 2 amounts were determined by ELISA using specific kits from R&D Systems, as instructed by the manufacturer.

2.7 NF- κ B activity assessment

TCa8113 and Tb cells were plated in 48-well plates, followed by transfection with a detection plasmid from the Cignal Reporter Assay kit (QIAGEN) after 24 hours. Each cell line was treated with experimental conditions the following day. Luciferase activity was assessed based on the Dual-Luciferase Reporter Assay System Protocol (Promega).

2.8 Clonogenic assays

Cells plated in 6-well plates were treated with experimental conditions of gemcitabine or gemcitabine combined with the lentivirus for 24 hours and 48 hours, respectively and re-plated in 6-well plates at the same density. Upon the colony formation, fixation with 4% formalin was performed followed by staining with a 0.5% crystal violet.

2.9 Transwell migration assays

Cells cultured in 10 cm dishes were administered with the experimental conditions of gemcitabine or gemcitabine combined with the lentivirus for 48 hours. The migration assay was carried out with a Transwell system containing 8 μ m-pore inserts (BD Biosciences, USA) according to the manufacturer’s instructions.

2.10 Animal studies

TCa8113 cells (1 × 10 7 ) in 100 μ L of PBS solution were administered to BALB/c nude mice subcutaneously in the abdominal region. The mice were randomly assigned to 5 groups ( n = 8), including the PBS, gemcitabine, NC + gemcitabine, shT1 + gemcitabine, and shT2 + gemcitabine groups. After the tumor volumes approximated 70–100 mm 3 , the intratumoral administration of PBS or 2 × 10 10 virus particles (VP) of lentiviruses in 50 μ l PBS was performed. This treatment was repeated three times and 50 μ l of gemcitabine (10 μ M) was co-administered on the day of the final lentiviral administration. After 7 days of various treatments, the effects on tumor growth and survival rates were measured and recorded.

2.11 Immunohistochemistry

Seven days after the final lentiviral administration, the tumor tissue samples were obtained and submitted to 10% formaldehyde fixation for more than 24 hours. IHC was carried out with anti-TGF- β 1 (Abcam), anti-TGF- β 2 (Abcam), and anti-ki67 primary antibodies. After deparaffinization with xylene and rehydration with an ethanol gradient, endogenous peroxidase was quenched by treatment with 0.1% H 2 O 2 . Then, a 10 mM citrate buffer (pH 6.0) was employed for antigen retrieval. Upon permeabilization (0.5% PBX) and blocking (5% BSA), the samples were incubated with primary antibodies overnight. HRP-conjugated secondary antibodies (Thermo Scientific) were employed for detection. Finally, DAB Plus Chromogen and DAB Plus Substrate (Thermo Fisher Scientific) were used for the color display.

2.12 Statistical analysis

Statistical analyses were performed with Graph Pad (Systat Software, USA). A P -value of 0.05 indicated statistical significance.

3.1 Gemcitabine induces changes in the survival and death-associated signaling pathways in OSCC

To explore the effects of gemcitabine in the cell cultures, we firstly determined the IC50 values of gemcitabine in various OSCC cell lines, which ranged between 2.5 μ M and 20 μ M (Fig. 1A). Then, key effectors in the survival and death signaling pathways were assessed, including PARP, p-p65, and p-AKT by immunoblot after the treatment with gemcitabine in the OSCC cell lines. The results showed that the phosphorylation levels of AKT and P65 were increased by the gemcitabine treatment while the cleaved form of PARP was not increased in a time-dependent manner (Fig. 1B). We also evaluated the cell viability after the treatment with gemcitabine by clonogenic assay and cell survival was high, even after the treatment with gemcitabine. These findings indicated the low effect of gemcitabine on OSCC, as well as the resistance of OSCC to gemcitabine treatment (Fig. 1C). Elevated p-p65 and p-Akt (Fig. 1B) levels suggested that the OSCC cells were gemcitabine resistant.

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After the administration of gemcitabine, the TGF- β levels in cell supernatants were determined by ELISA. As shown in Fig. 2A, gemcitabine increased the TGF- β 1 expression in a time-dependent manner. TGF- β 1 and TGF- β 2 expressions were also dramatically elevated in gemcitabine resistant cells, which were the cells that were still alive after incubation of gemcitabine at IC50 (Fig. 2B).

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Cell viability was also significantly reduced by the treatment with the TGF- β protein (Fig. 2C). We designed a lentivirus harboring the shRNA of TGF- β 1/2, which can downregulate TGF- β 1/2 (Fig. 2D).

As shown in Fig. 3A, cell viability was effectively reduced in the combination group compared with the NC virus-infected group. NF-kB activity was also decreased dramatically in the combination treatment group but increased by gemcitabine when used alone (Fig. 3B). Subsequently, we screened various signaling pathway molecules, including PARP, p65, and AKT. We observed that the p-AKT and p-P65 amounts were elevated after treatment with gemcitabine alone but decreased in the combination group; meanwhile, cleaved PARP levels were effectively increased (Fig. 3C). The emergence of clones originated from TCA8113 was reduced dramatically in the combination group (Fig. 3D).

3.4 The co-treatment of gemcitabine and the lentivirus-expressing shRNA against TGF- β could suppress EMT and migration

The EMT includes physiological and pathophysiological events in which epithelial cells are converted into mesenchymal cells for functional needs. In cancer, the EMT contributes to the early-stage dissemination of cancer cells followed by invasion and metastasis, as well as drug resistance.

As shown in Fig. 4A, the exposure of TCA8113 and Tb cells to gemcitabine caused the upregulation of the mesenchymal marker vimentin while markedly decreasing epithelial marker E-cadherin levels. However, the gemcitabine-induced regulation of EMT markers was impaired by pretreatment with the lentivirus downregulating the expression of TGF- β .

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Moreover, Transwell migration assays demonstrated that the amounts of migrated cells after TGF- β downregulation were reduced in comparison with the gemcitabine resistance groups ( 0.001) (Fig. 4B).

Since the abovementioned lentivirus expressing shTGF- β 1/2 enhanced apoptosis and decreased TGF- β 1/2 levels, thereby overcoming TCA8113 cell resistance to gemcitabine, we designed an in vivo experiment to further assess the anti-tumor effects of this therapeutic combination in xenograft animal models. Whether the combination could overcome TCA8113 cell resistance to gemcitabine was determined.

The results showed that treatment with gemcitabine followed by the lentivirus expressing shTGF- β 1/2 exerted enhanced anti-tumor effects in comparison with gemcitabine as monotherapy (Fig. 5A). However, the resistance was not entirely abolished. Animal survival was highest after administering the combined therapy with the lentivirus expressing shTGF- β 1/2 and gemcitabine with no mice dying until 19 days post-treatment (Fig. 5B). At this point, the gemcitabine alone and PBS groups displayed survival rates of 40% and 0%, respectively (Fig. 5B).

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The expression levels of Ki-67 were also markedly elevated in the gemcitabine alone group and reduced in the combination group. The staining index of Ki-67 after administering the combined treatment was significantly reduced in comparison to the resistance group.

As shown in the ex vivo experiments, combined treatment with the lentivirus expressing shTGF- β 1/2 and gemcitabine noticeably increased the anti-tumor effects while effectively overriding gemcitabine resistance in animal models of oral cancer.