Background
Oral squamous cell carcinoma (OSCC) is the most common oral cancer; it is characterized by a high degree of local invasiveness and proliferation capacity and a relatively poor prognosis [
1‐
3]. Among OSCCs, tongue squamous cell cancer (TSCC) has the highest incidence and is often associated with a low survival rate [
4,
5]. TSCC also results in the highest rates of mortality among head and neck region cancers [
6,
7]. Thus, it is imperative to explore novel agents or treatment strategies targeting TSCC.
Tian-Hua-Fen (
Trichosanthes kirilowii Maxim.) is a plant renowned in traditional Chinese medicine and is used as an abortifacient based on its directed toxicity toward trophoblasts and choriocarcinoma cells [
8‐
11]. Trichosanthin (TCS), extracted from the root tubers of Tian-Hua-Fen, is a type 1 ribosome-inactivating protein (RIP). It has been reported that treating tumor cells with TCS causes cell death by inducing cell necrosis and inhibiting cellular protein synthesis [
12]. Recently, a series of studies have revealed the antitumor effects of TCS, suggesting it has apoptotic activities in numerous types of tumors, including breast cancer, nasopharyngeal carcinoma, hepatocellular carcinoma, non-small cell lung cancer, cervical cancer, and B-cell lymphoma [
11,
13‐
18]. Therefore, TCS represents a potential novel therapeutic drug for antitumor treatment.
As a major constituent involved in cytotoxic T lymphocyte (CTL)-mediated tumor cell apoptosis, the mechanism of granzyme B (GrzB)-mediated cell death has been reasonably well defined [
19,
20]. In a previous study, our group reported that TCS increases GrzB penetration of tumor cells by upregulating the cation-independent mannose-6-phosphate receptor (CI-MPR) [
17]. These observations suggested that TCS combined with GrzB might be a superior approach to enhance the efficacy of cancer immunotherapy [
17]. Nevertheless, the antitumor effects of TCS and GrzB in TSCC remain largely unknown. The present study aimed to evaluate the potential antitumor activity of TCS or GrzB alone versus TCS and GrzB combined. Further studies were also conducted to explore the molecular mechanisms regulating TCS and/or GrzB targeting TSCC.
Methods
Cell culture
Cells of the human squamous cell carcinoma lines SCC15 and SCC25 were purchased from the American Type Culture Collection and cytogenetically tested and authenticated prior to freezing. The two cell lines were routinely passaged in a 1:1 mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium (Gibco, Thermo) containing 1.2 g/L sodium bicarbonate, 2.5 mM L-glutamine, 15 mM HEPES, and 0.5 mM sodium pyruvate supplemented with 400 ng/mL hydrocortisone, 90% fetal bovine serum (Gibco, Thermo), 10% 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 incubator.
Xenograft tumor model
All animal experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University Shenzhen Graduate School. Eight-week-old male BALB/c nude mice (Guangdong Medical Laboratory Animal Center, Guangzhou) were maintained under specific pathogen-free (SPF) conditions and were free to access sterilized food pellets and distilled water, with a 12-h light/dark cycle. Each mouse was subcutaneously inoculated with 2 × 106 SCC25 cells in the right hind-limb. Three days later, tumor formation was assessed in each mouse for further drug treatment.
Trichosanthin and granzyme B treatment
The SCC25 tumor-bearing mice were randomly divided into four groups: phosphate-buffered saline (PBS) control group, TCS group, GrzB group, and TCS/GrzB combination group (n = 8–9 per group). TCS protein was extracted in our laboratory, while recombinant granzyme B (active) was purchased from Sino Biological. TCS (2 μg/g body weight) was diluted in 60 μL PBS and intraperitoneally injected on days 1, 3, 5, and 7 in the TCS and combination groups. Active GrzB (2 μg/100 g body weight) was diluted in 60 μL PBS and intraperitoneally injected on days 2, 4, 6, and 8 in the GrzB and combination groups. Mice in the PBS group were intraperitoneally injected with 60 μl PBS each day during the administration period. Tumors were assessed every other day using Vernier calipers, and tumor volumes were calculated using the elliptical formula: 1/2 (long diameter × short diameter2). On day 16, all mice were sacrificed by cervical dislocation, and tumors were weighed after being separated from the surrounding muscle and dermis. For each group, tumor tissues were collected and divided into three samples: the first sample was homogenized into tumor lysis for western blot analysis, the second sample was fixed with 10% neutral formalin and embedded in paraffin for hematoxylin and eosin (HE) staining and immunofluorescence analysis, and the third sample was stored in liquid nitrogen for RNA extraction. The tumor inhibition rate was calculated using the following equation: Tumor inhibition rate (%) = (1 − average volume of experimental group / average volume of control group) × 100%.
Hematoxylin and eosin staining and morphological analysis
The fresh tumor tissues were washed with normal saline then placed in 10% neutral formalin solution and stored overnight at 4 °C. The next day, the fixed samples were dehydrated, embedded in paraffin, and sectioned at 6-μm thickness using a Leica microtome. After drying overnight, the slides were stained with H&E according to the standard protocol [
21]. The stained sections were dehydrated with ethanol, rendered transparent with xylene, and sealed. The tissue morphology profiles were observed under an optical microscope (Olympus, Japan).
Western blot analysis
Total protein was extracted by lysing fresh tumor tissues in precooling RIPA buffer (Thermo), supplemented with protease inhibitor cocktail (MCE) and PhosSTOP™ phosphatase inhibitor (Roche), quantitated using a Pierce™ BCA Protein Assay (Thermo) and transferred onto a PVDF membrane (Millipore) after separation by SDS-PAGE. The membranes were incubated with respective primary antibodies at 4 °C overnight then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody, as previously described [
17]. Chemiluminescence was developed using ECL Ultra HRP substrate (Merck) and photographed under the SAGECREATION ChemiMini™ Imaging System.
Quantitative real-time PCR
Total RNA was isolated from tumor tissues using the Eastep™ Super Total RNA Extraction Kit (Promega) and reverse transcribed into cDNA using the PrimeScript™ RT Reagent kit (TaKaRa), according to the manufacturer’s instructions. The synthesized cDNA was then amplified by quantitative PCR using TB Green Premix Ex Taq (TaKaRa) on a Quantstudio™ 7 Flex Real-Time PCR System (ABI). The expression of target genes was normalized against GAPDH using the 2
-ΔΔCt assay. Primer oligos were synthesized by TSINGKE Biological Co., Ltd., (Beijing, China) and are listed in Table
1.
P53 | P53-qPCR-F | TAGTGTGGTGGTGCCCTATG |
P53-qPCR-R | CCAGTGTGATGATGGTGAGG |
BAD | Bad-qPCR-F | TACCTGCCTCTGCCTTCCA |
Bad-qPCR-R | CTGCTCACTCGGCTCAAACT |
VEGFA | VEGFA-qPCR-F | AGGGCAGAATCATCACGAAGT |
VEGFA-qPCR-R | AGGGTCTCGATTGGATGGCA |
GAPDH | gapdh-qPCR-F | GTCAACGGATTTGGTCGTATTG |
gapdh-qPCR-R | CATGGGTGGAATCATATTGGAA |
Histology and immunofluorescence
The paraffin-embedded tissue sections (6 μm) were deparaffinized by immersion in fresh xylene and hydrated by immersion in graded ethanol washes. The sections were then immersed in citrate buffer (pH 6.0) at 95 °C for 20 min for antigen retrieval before being incubated with 3% donkey serum at room temperature for 30 min. The slides were then incubated with one of the following primary antibodies: monoclonal rabbit anti-human cleaved caspase-3 (clone 5A1E, Cell Signaling Tech.) at 1:100; monoclonal rabbit anti-human PCNA (clone D3H8P, Cell Signaling Tech.) at 1:100; monoclonal rabbit anti-human Ki67 (Cell Signaling Tech.) at 1:2000; monoclonal rabbit anti-human caspase-7 (Cell Signaling Tech.) at 1:200, or monoclonal mouse anti-human Bcl2 (sc-7382, Santa Cruz) at 1:100 overnight at 4 °C in a humidified box. The next day, after washing in PBST, the sections were incubated with Alexa Fluor 488 or Alexa Fluor 555 goat anti-mouse secondary Ab (Thermo) (1:200 dilution) for 2 h at room temperature in a humidified box in the dark. Next, nuclear staining was performed with 4′,6-diamidino-2-phenylindole (DAPI) (Cell Signaling Tech.). Finally, the fluorescence was observed and images were taken under a FV3000 confocal microscope (OLYMPUS, Japan).
TUNEL staining assay
For the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay, procedures were performed using the DeadEnd™ Fluorometric TUNEL System (Promega, USA). After deparaffinizing and hydrating, the staining procedures were carried out according to the manufacturer’s instructions. Sections were stained with DAPI and mounted using ProLong® Gold Antifade Reagent (Cell Signaling Tech.). The nuclei-stained green was an apoptotic cell. The Bcl-2/Bcl-xL inhibitor, ABT-263 (8 μM; Selleckchem, Houston, TX, USA), was used as a positive control. Apoptosis was inhibited using Z-VAD-FMK (pan-caspase inhibitor). Three fields were chosen at random and analyzed for positive signaling. The apoptotic index was calculated as the percentage of positive-stained cells: number of apoptotic cells (green)/total number of nucleated cells.
Statistical analysis
Data (mean ± SEM, 2–3 experiments) were analyzed for statistical significance using a t-test. A p-value < 0.05 was considered statistically significant. GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used to perform the statistical analyses.
Discussion
Tongue squamous cell cancer has the highest incidence among all oral cancers, with an approximately 60% 5-year survival rate following comprehensive sequential therapies [
4]. Furthermore, TSCC has the highest rates of mortality among head and neck region cancers, constituting a major challenge for head and neck surgeons [
3,
6,
7]. The tongue is a highly vascularized and mobile organ, making it impossible to surgically eradicate tongue tumors without tumor cells migrating along circulation [
29,
30]
. In clinical practice, pre- and/or post-surgery chemotherapy or radiotherapy for tongue cancer is always necessary [
31‐
34]. However, the drugs used for TSCC chemotherapy have limited efficacy and unsatisfactory clinical outcomes [
35,
36]. In recent years, several new drugs that target tongue cancer cells have been reported [
37,
38]. We recently reported that TCS combined with GrzB might be a superior treatment for enhancing the efficacy of liver cancer immunotherapy [
17]. In the present study, we investigated the antitumor efficacy of TCS and GrzB combination therapy on TSCC.
TCS is used as an anti-inflammatory agent in traditional Chinese medicine [
9,
39]. Recently, TCS has been investigated for its antitumor activity against several types of tumors, based on its ability to induce apoptosis [
9,
11,
18,
40‐
42]. However, TCS was found to exhibit antigenicity and cytotoxicity at high dosages, hindering its clinical applications [
43]. In our previous study, we found that low a dose of TCS significantly increased cell surface expression of CI-MPR, a cell-death receptor for GrzB protein transportation during cytotoxic T cell-induced apoptosis [
17]. In the present study, we first screened the potential antitumor activity of TCS using two types of SCC cell lines and confirmed that the SCC25 line exhibited greater sensitivity to TCS. TCS also resulted in significantly decreased viability of SCC cells at lower concentrations compared with the reduction in viability obtained using the traditional chemotherapy drug, cisplatin. To investigate the effect of TCS and/or GrzB on TSCC in vivo, a xenograft SCC25 tumor model was established and validated, following a previously established method [
37,
38], in which TSCC tumors form 3 days following a subcutaneous injection of a TSCC cell suspension. After 16 days of administration of TCS, GrzB, or a TCS/GrzB combination, the tumors were excised and collected for histology, immunohistology, and immunoblot analyses. It was found that, compared with tumor growth in the control group, TCS or GrzB treatment significantly inhibited tumor growth. TCS combined with GrzB treatment led to a more pronounced inhibition of tumor growth compared with the inhibition resulting from treatment with either drug alone. These results suggest that the TCS and GrzB combination treatment has more potent antitumor activity toward tongue cancers in an in vivo mouse model.
Previous studies have revealed that TCS-induced apoptosis is associated with caspase activation [
11,
15,
16]. Caspases are principal effectors that play critical roles in the induction of apoptosis, cleaving targets to execute cell death [
44,
45]. Caspase-3 and caspase-7 have long been recognized as the key proteases in the cell demolition processes involved in apoptosis, through the targeting of structural substrates including cell–cell adherence junctions, focal adhesion sites, and nuclear laminins [
46]. These two caspase proteases function in a similar way during the execution phase of apoptosis. It has been reported that caspase-3-deficient cells can continue apoptosis if caspase-7 is present [
47]. However, apoptosis cannot occur in cells lacking both caspase-3 and caspase-7 [
48]. In the present study, cleavage of both caspase-3 and caspase-7 was observed in the TCS and GrzB-treated groups, indicating both caspase-3 and caspase-7 take part in apoptosis.
The development of oral cancer is a complicated process, driven by multiple genes and involving multiple steps. Generally, the hallmarks of cancer comprise six biological processes: sustaining proliferative signaling, evading growth suppressors, resisting cell death, enabling replicative immortality, inducing angiogenesis, and activating invasion and metastasis [
49]. To mechanistically explain our observations, we also investigated the effect of TCS treatment on markers of tumor proliferation (Ki67, PCNA). Our present study demonstrated that TCS and GrzB decreased cell proliferation levels. Targeting angiogenesis to prevent tumor progression is a novel direction for cancer therapy [
50]. In a previous study, He et al reported that VEGF expression and secretion were significantly decreased in JAR cells following treatment with TCS [
51]. Here, we observed a decrease in the expression of angiogenesis marker (VEGF-A) in tumor tissue extracted from the TCS/GrzB treatment group. These results suggest that the VEGF pathway is involved in the anti-angiogenic effects of TCS and GrzB.
Conclusion
The combination of TCS and GrzB treatment exhibited superior inhibitory effects on SCC25 tumor growth in vivo. TCS and GrzB suppressed SCC cell proliferation by downregulating Ki67, PCNA, VEGF-A, and Bcl2 expression, while accelerating apoptosis via the upregulation of cleaved caspase-3, caspase-7, and BAD activity. The combination of TCS and GrzB could represent a more potent immunotherapeutic protocol for the treatment of oral squamous cell carcinoma, and further investigations are warranted.
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