Research ArticleExperimental Studies

Study of the Acquisition of Sensitivity to Paclitaxel Plus Cetuximab by the Addition of Low-dose Proteasome Inhibitor

MASAKI YODA, KANAME SAKUMA, TOMOYUKI KII and AKIRA TANAKA
Anticancer Research September 2022, 42 (9) 4273-4283; DOI: https://doi.org/10.21873/anticanres.15927

Abstract

Background/Aim: Although paclitaxel plus cetuximab for recurrent/metastatic oral squamous cell carcinoma (OSCC) has a relatively high success rate, many cases are refractory. We investigated the change in nuclear factor-kappa B (NF-Embedded ImageB) expression after this combination therapy using microcollagen 3D cell culture. We also investigated changes in antitumor efficacy using low doses of paclitaxel-cetuximab combined with the proteasome inhibitor bortezomib on a cell line with low sensitivity to paclitaxel plus cetuximab. Materials and Methods: Eight human OSCC cell lines were cultured in 3D and exposed to paclitaxel-cetuximab. real-time polymerase chain reaction was used to evaluate NF-Embedded ImageB mRNA expression in OSCC cell lines in vivo and in vitro after exposure to anticancer agents. Activity at the protein level was confirmed using western blotting. Bortezomib (0.002-0.4 μg/ml) was added to paclitaxel-cetuximab and its effects assessed in OSCC cell lines with low paclitaxel-cetuximab sensitivity. Results: mRNA and protein expression of NF-Embedded ImageB was significantly reduced after treatment with paclitaxel-cetuximab in cell lines sensitive to this combination. In contrast, both mRNA and protein expression significantly increased in the cell lines with low sensitivity to paclitaxel plus cetuximab. The addition of low concentrations of bortezomib to cell lines with low sensitivity to paclitaxel-cetuximab was found to enhance antitumor efficacy. Conclusion: Increased NF-Embedded ImageB expression strongly contributes to resistance to paclitaxel-cetuximab, suggesting that the administration of small doses of bortezomib, which inhibits NF-Embedded ImageB, combined with paclitaxel-cetuximab may enhance antitumor efficacy against cancer cells with low sensitivity to the combination therapy.

Key Words:

In addition to conventional platinum-based drugs, notable advances have been made in chemotherapy for recurrent or metastasized oral carcinoma, including molecular targeted therapies and immune checkpoint inhibitors (1). Among these, the combined administration of paclitaxel and cetuximab has been reported to have a high success rate, ranging from 38% to 55% (2, 3). This combination therapy can also be used in patients who have disease resistant to platinum-based drugs or who have severe kidney dysfunction, which is a serious adverse reaction to platinum-based drugs (4); thus, it is an important third-line therapy for recurrent, metastasized head and neck squamous cell carcinoma. Although a small number of studies have reported that genetic change is the primary cause of efficacy enhancement and resistance resulting from the combined administration of paclitaxel-cetuximab (5, 6), the basic mechanism remains unknown.

Epidermal growth factor receptor (EGFR) is strongly involved in tumor proliferation and molecular targeted therapy in oral cancer (7, 8). Nuclear factor-kappa B (NF-Embedded ImageB), which is expressed downstream of the EGFR signal transmission pathway, promotes cancer cell proliferation and chemotherapy resistance by increasing NF-Embedded ImageB activity (9-11). The mechanism of the enhanced antitumor efficacy of paclitaxel-cetuximab has been reported to be NF-Embedded ImageB activation by paclitaxel (6, 12) and a synergistic reduction in NF-Embedded ImageB expression that results from the combined use of cetuximab (6). However, these reports only confirmed genetic changes after contact with paclitaxel-cetuximab in monolayer cultures of OSCC cell lines, and there are few reports confirming these changes actually occur in long-term in vitro micro 3D culture.

It is difficult to investigate the genetic changes that occur after the administration of anticancer drugs in vivo and in clinical specimens. Therefore, in the present study, we used the collagen gel droplet-embedded culture drug sensitivity test (CD-DST), a microcollagen 3D culture treatment, to confirm the sequential changes that NF-Embedded ImageB undergoes as a result of exposure to paclitaxel-cetuximab. CD-DST can determine the antitumor sensitivity for multiple drugs, including molecular targeted drugs. In addition, appropriate concentrations of antitumor drugs for experimental models using oral carcinoma cells have already been established (13-15). CD-DST also has in vivo reproducibility, and because microcollagen 3D cultivation allows for long-term cultivation, the confirmation of sequential genetic changes is possible (5, 15, 16). Therefore, in this study, we determined the expression of NF-Embedded ImageB in oral squamous cell carcinoma (OSCC) cell lines after exposure to paclitaxel-cetuximab and investigated their sensitivity to determine resistance mechanisms. Furthermore, in OSCC cell lines with low sensitivity after exposure to paclitaxel-cetuximab, we investigated the restoration of sensitivity via the combined use of the proteasome inhibitor bortezomib, which inhibits NF-Embedded ImageB.

Materials and Methods

Materials. Eight OSCC cell lines were used in this study: SAS, HSC-3, HSC-4, and OSC-20, squamous cell carcinoma of the tongue; HSC-2, oral cavity squamous cell carcinoma; HO-1-u-1, floor of the mouth squamous cell carcinoma; KON, floor of the mouth squamous cell carcinoma; and SAT, OSCC. The OSCC cell lines were purchased from the National Institutes of Biomedical Innovation, Health and Nutrition, Japanese Collection of Research Bioresources Cell Bank (Osaka, Japan). These cell lines were passaged in an incubator at 37°C, with 95% humidity, and 5% CO2 in plastic Petri dishes with a diameter of 35 mm. The culture medium used was Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (Nihon Pharmaceutical Co., Ltd., Tokyo, Japan), to which 10% fetal bovine serum (FBS; Life Technologies, Van Allen Way, CA, USA), 0.1% DMEM nonessential amino acids solution (Life Technologies), 1% penicillin–streptomycin (Life Technologies) and 0.1% fungizone (Life Technologies) were added. The anticancer drugs used were cetuximab (Erbitux® injection solution, 100 mg/20 ml; Merck Serono, Tokyo, Japan), paclitaxel (Paclitaxel Injectable®, 100 mg/16.7 ml; Nippon Kayaku, Tokyo, Japan) and bortezomib (Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan). We also used 5-week-old female nude mice, BALB/c/nu/nu (Japan Charles River Co., Ltd., Tokyo, Japan), which were raised under specific pathogen-free conditions, for in vivo study.

Assessment of sensitivity of OSCC cell lines to paclitaxel, cetuximab and paclitaxel-cetuximab using CD-DST. We used a primary human cancer cell culture kit Primaster® (Kurabo Industries, Ltd., Osaka, Japan), which uses the method reported by Kobayashi et al. (17) for CD-DST. This kit includes the cell-dispersant enzyme EZ, collagen gel (CG) flasks, preculture medium (PCM)-1, collagen solution (solution A), 10× F-12 culture medium (solution B), reconstitution buffer (solution C), serum-free medium PCM-2, neutral red solution and a collagen drop culture kit. T The OSCC cell lines (eight lines) recovered from the collagen kid were cultured with the mix solution in a volume ratio as follows: solution A:solution B:solution C=8:1:1 and adjusted to 2×105 to 5×105 cells/ml. The adjusted CG cell suspension solution was transferred to a six-well Petri dish using a micropipette, with three drops of 30 μl each per well. The dish was then placed in an incubator at 37°C/5% CO2 for 1 h to allow for gelation before being cultured for 24 h in 10% FBS-supplemented DMEM/F12 culture medium. In accordance with the method used by Kii et al. (15), cells were treated with 0.1 μg/ml paclitaxel for 24 h, then with 250 μg/ml cetuximab for 144 h according to Ryuki et al. (14). Following drug exposure, the cultures were washed with phosphate-buffered saline, transferred to PCM-2 serum-free culture medium and cultured for a total of 6 days. After completion of the culturing process, neutral red solution was added to the cells. After staining for 2 h in an incubator at 37°C/5% CO2, the cultures were fixed for 40 min in 10% neutral formalin solution. After fixation, the samples were washed with water and dried to prepare study samples. Anticancer efficacy was evaluated using a Primage® image analyzer (Kurabo Industries, Ltd.) according to the method described by Koezuka et al. (18). The growth of cancer cells was confirmed, and the antitumor effects were determined by measuring the colony volume based on the images of cancer cells. The ratio of the volume in the cells exposed to anticancer drugs (T: Treatment) to the volume in control (C: control) cells was used to determine the T/C value, and was taken as representing the ratio of the proliferation rate of treated and untreated tumor cells; OSSC cell lines were classified as having high or low sensitivity using the criteria of T/C ≤50% and T/C >50%, respectively. These assessment criteria allowed us to evaluate rates of tumor cell proliferation that were at least 0.8 times the rate at the start of the trial (Figure 1).

Figure 1.
Figure 1.

Overview of the collagen gel droplet-embedded culture drug sensitivity test (CD-DST). We confirmed sensitivity to paclitaxel (PTX), cetuximab (Cmab) and their combination for eight oral squamous cell carcinoma cell lines.

Reverse transcription polymerase chain reaction (RT-PCR) for determination of gene expression of NF-Embedded ImageB. Total RNA was extracted from the using ISOGEN II (Nippon Gene, Tokyo, Japan) according to the manufacturer’s protocol. Using 1 μg of the extracted total RNA, cDNA was synthesized using a high-capacity cDNA reverse transcription kit (Life Technologies). After conducting a RT-PCR cycle at 25°C for 10 min, 37°C for 60 min, 37°C for 60 min and 85°C for 5 min, Platinum PCR Super Mix (Life Technologies) and NF-Embedded ImageB primers were used to perform PCR amplification (denaturation: 94°C for 30 s; annealing: 55°C for 30 s; and extension: 72°C for 60 s) for 35 cycles (Table I). PCR amplification was performed under the same conditions using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the internal control. The amplified PCR products were subjected to electrophoresis using a 2% agarose gel (Nippon Gene), visualized using ethidium bromide and confirmed using an ultraviolet (UV) irradiation gel imaging device.

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Table I.

Primer sequence and polymerase chain reaction conditions.

Quantitative comparisons of the expression levels of NF-Embedded ImageB using real-time PCR. Expression in vitro: Using real-time PCR, we perform quantitative comparisons of the mRNA expression levels of NF-Embedded ImageB. The eight OSCC cell lines were 3D-cultured in collagen gel and treated as described above; the control was not exposed to anticancer drugs. CG was then dissolved the in the cell dispersant enzyme EZ and the cells were recovered. Following cell harvesting, total RNA was extracted using the ISOGEN II protocol (Nippon gene, Tokyo, Japan). To perform quantitative comparisons of the NF-Embedded ImageB expression levels, we performed real-time PCR using PrimeScript RT™ reagent kit with gDNA Eraser (Perfect Real Time, TAKARA BIO Inc., Shiga, Japan) according to the manufacturer’s protocol.

In vivo tumor effect. We used five OSCC lines (1×107 cells; HSC-2, OSC-20, SAS, SAT and HSC-4) suspended in 0.5 ml Hank’s solution before subcutaneous injection into the backs of three nude mice per cell line using 23G Terumo Syringes®. Subsequently, when the tumor size (1/2× major axis × minor axis2) reached 100 to 150 mm3, we initiated intra-abdominal administration of paclitaxel (20 mg/kg/day, twice/week) with/without cetuximab (20 mg/kg/day, twice/week) for 3 weeks (6). Twenty-one days after the start of anticancer drug administration, the nude mice were euthanized using intra-abdominal injections of 200 mg/kg sodium pentobarbital, and the tumors were excised and homogenized using BioMasher® III. The total RNA was then extracted using ISOGEN II (Nippon Gene) according to the manufacturer’s protocol. Determination of the in vivo expression of NF-Embedded ImageB was performed by real-time PCR as described above. The animal experiments performed as part of this study were approved by the Animal Experiment Institutional Review Board of the Nippon Dental University School of Life Dentistry (approval no. ECNG-H-131).

Comparison of the expression of NF-Embedded ImageB protein using western blot analysis. Western blot analysis was used to compare the expression of NF-Embedded ImageB protein in cell lines with different drug sensitivity. We applied CD-DST. CG was dissolved using the cell dispersant enzyme EZ, and the cells were recovered. Subsequently, total protein was extracted using RIPA buffer in accordance with the prescribed protocol and subjected to 12.5% sodium docedyl sulfate–polyacrylamide gel electrophoresis to break down the extracted protein. Using a minislab electrophoresis device (Atto Co., Ltd., Tokyo, Japan), the proteins were transferred from the gel to a polyvinylidene difluoride membrane and exposed to antibodies for western blotting. Primary antibody to NF-Embedded ImageB P65 [rabbit monoclonal (E379) to NF-Embedded ImageB P65], and anti-rabbit immunoglobulin G horseradish peroxidase conjugated secondary antibody (Promega Corporation, Madison, WI, USA) and membranes were then processed using the iBind™ Western System (Life Technologies Japan Ltd., Tokyo, Japan). Qualitative detection was performed using UV.

Confirmation of changes in sensitivity to bortezomib with CD-DST application. Using the three lines (SAT, HSC-4 and KON) that were found via CD-DST to have low sensitivity to paclitaxel-cetuximab, we set the concentration in five steps with reference to the maximum concentration in blood (Cmax) (0.002, 0.02, 0.1, 0.2 and 0.4 μg/ml) using the test concentration as the criterion (0.2 μg/ml) and bortezomib Cmax (0.223 μg/ml) as the reference (19).Treatment with bortezomib lasted for 24 h. Using the standard method, a quantitative comparison was made after CD-DST was performed on the following three groups: Control (no anticancer drug) group, bortezomib monotherapy, and the paclitaxel-cetuximab plus bortezomib combination therapy.

Statistical analysis. The RT-PCR results were normalized based on GAPDH expression, and the percentage of change compared with the results of the untreated control was determined. Expression was measured in each OSCC cell line six times. We used the ΔΔCT method for analysis and one-way analysis of variance to compare NF-Embedded ImageB levels. The statistical analysis software used was the Bell Curve for Excel version 3.20 (Social Survey Research Information, Tokyo, Japan).

Results

Assessment of the sensitivity of eight OSCC cell lines to paclitaxel, cetuximab and paclitaxel-cetuximab using CD-DST. Using CD-DST, we evaluated drug sensitivity using the following criteria: T/C ≤50% was considered high sensitivity, whereas T/C ≤50% was considered low sensitivity. HSC-2, HO-1-u-1 and OSC-20 were found to be in the group with high sensitivity to paclitaxel-cetuximab; SAS and HSC-3 were highly sensitive to paclitaxel monotherapy, and paclitaxel-cetuximab combination therapy; while SAT, HSC-4 and KON had low sensitivity to paclitaxel-cetuximab (Table II).

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Table II.

Assessment of sensitivity of oral squamous cell carcinoma cell lines treated with paclitaxel (PTX), cetuximab (Cmab) and their combination using collagen gel droplet-embedded culture drug sensitivity test (CD-DST). The cell volumes from image analysis of cell lines in 3D culture were determined and cell lines were considered to have high sensitivity when the ratio of the cell volume T (treatment)/C (control) was ≤50% (shown in bold) and low sensitivity when T/C was >50%.

Expression of NF-Embedded ImageB mRNA in vitro. All eight OSCC cell lines were confirmed to express NF-Embedded ImageB mRNA (Figure 2). We conducted a quantitative comparison of the levels of NF-Embedded ImageB gene expression after paclitaxel monotherapy, cetuximab monotherapy and paclitaxel-cetuximab treatment. We found no significant change in NF-Embedded ImageB expression as a result of either monotherapy in the cells with high sensitivity to paclitaxel-cetuximab (HSC-2, HO-1-u-1 and OSC-20), nor in those with high sensitivity to paclitaxel monotherapy and paclitaxel-cetuximab (SAS and HSC-3). However, expression of NF-Embedded ImageB was significantly reduced as a result of exposure to the paclitaxel-cetuximab combination in these cells lines (Figure 3). Monotherapies also failed to induce a significant change in NF-Embedded ImageB expression in the cell lines with low paclitaxel-cetuximab sensitivity, SAT, HSC-4 and KON; however, we noted a significant increase in expression of NF-Embedded ImageB mRNA in these cell lines after exposure to paclitaxel-cetuximab.

Figure 2.
Figure 2.

Confirmation of expression of nuclear factor-kappa B (NF-Embedded ImageB) gene using real-time polymerase chain reaction. From the right: HSC-2, HO-1-u-1, OSC-20, SAS, HSC-3, SAT, HSC-4 and KON cells lines. All eight lines showed NF-Embedded ImageB expression.

Figure 3.
Figure 3.

In vitro mRNA expression of nuclear factor-kappa B (NF-Embedded ImageB) after treatment with anticancer drugs. The level of NF-Embedded ImageB expression in cell lines showing high sensitivity to paclitaxel (PTX) plus cetuximab (Cmab) was significantly reduced after treatment with paclitaxel-cetuximab; however, expression in cell lines with low sensitivity increased. Data are plotted as the mean±standard deviation (n=6) and analyzed using one-way analysis of variance. Significantly different at *p<0.05 and **p<0.01.

NF-Embedded ImageB mRNA expression in vivo. We quantitatively compared the mRNA levels of NF-Embedded ImageB expression after paclitaxel monotherapy, cetuximab monotherapy and paclitaxel-cetuximab treatment in mice bearing xenografts from five cell lines: HSC-2 and OSC-20 (high sensitivity to paclitaxel-cetuximab), SAS (high sensitivity to paclitaxel monotherapy and paclitaxel-cetuximab combination therapy) and SAT and HSC-4 (low sensitivity to paclitaxel-cetuximab). Similar to the in vitro results, we found that paclitaxel-cetuximab caused a significant decrease in the NF-Embedded ImageB expression in the HSC-2 and OSC-20 tumors as well as in SAS, cell lines with high sensitivity to the combination therapy. In addition, we found a significant increase in NF-Embedded ImageB expression resulting from exposure to paclitaxel alone, cetuximab alone and paclitaxel-cetuximab in SAT and HSC-4 cell lines with low paclitaxel-cetuximab sensitivity (Figure 4).

Figure 4.
Figure 4.

In vivo mRNA expression of nuclear factor-kappa B (NF-Embedded ImageB) after the administration of anticancer drugs. Using nude mice, tumors were derived from cell lines (1×107 cells; HSC-2, OSC-20, SAS, SAT, and HSC-4) and mice were assigned to an untreated control group, a paclitaxel (PTX) monotherapy group, a cetuximab (Cmab) monotherapy group and a paclitaxel-cetuximab group. We then compared the level of NF-Embedded ImageB expression in tumors. In line with our in vitro experiments, tumors from cell lines with low sensitivity to paclitaxel-cetuximab were found to have increased NF-Embedded ImageB expression following exposure to paclitaxel-cetuximab. Data are plotted as mean±standard deviation (n=6) and analyzed using one-way analysis of variance. Significantly different at *p<0.05 and **p<0.01.

Comparison of NF-Embedded ImageB protein expression using western blot analysis. We confirmed NF-Embedded ImageB protein expression for the eight cell lines. NF-Embedded ImageB protein expression was found to be suppressed following exposure to paclitaxel-cetuximab in cell line with high sensitivity to this combination. In contrast, in the cell lines with low sensitivity, we observed an increase in NF-Embedded ImageB protein as a result of paclitaxel-cetuximab therapy (Figure 5).

Figure 5.
Figure 5.

Comparison of nuclear factor-kappa B (NF-Embedded ImageB) protein expression in oral squamous cell carcinoma cell lines using western blot analysis after treatment with paclitaxel (PTX) and cetuximab (Cmab), alone and in combination. Following treatment, the level of NF-Embedded ImageB expression was found to be notably reduced in the lines with high sensitivity to paclitaxel-cetuximab; however, its expression was notably increased in the lines with low sensitivity.

Assessment of sensitivity to the combined use of bortezomib via CD-DST. We found a concentration-dependent antitumor effect with both bortezomib monotherapy and combined use. In addition, with the addition of bortezomib at 1/10 (0.02 μg/ml) Cmax (0.2 μg/ml), we found enhanced antitumor effects and high-sensitivity after contact with paclitaxel-cetuximab (Figure 6; Table III).

Figure 6.
Figure 6.

Assessment of the sensitivity to bortezomib monotherapy and combined therapy when used on three cell lines (SAT, HSC-4 and KON) with low sensitivity to paclitaxel-cetuximab (PTX-Cmab). The results showed concentration dependency for both bortezomib monotherapy and combination therapy and an increased antitumor effect when a dose as low as 0.02 μg/ml bortezomib was combined with paclitaxel-cetuximab.

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Table III.

Assessment of the sensitivity to bortezomib monotherapy at five concentrations using collagen gel droplet-embedded culture drug sensitivity test (CD-DST) in cell lines insensitive to paclitaxel-cetuximab therapy. The cell volumes from image analysis of cell lines in 3D culture were determined and cell lines were considered to have high sensitivity when the ratio T (treatment)/C (control) was ≤50% (shown in bold) and low sensitivity when T/C was >50%.

Discussion

The combination of paclitaxel and cetuximab is used as first-line treatment for recurrent/metastatic head and neck cancer (20, 21). Nevertheless, major issues include the fact that to prevent kidney dysfunction, pre-/posthydration via in-hospital treatment is required and not indicated for patients with pre-existing kidney dysfunction (22). In addition, the programmed cell death protein 1 (PD1) antibody nivolumab was approved as a secondary treatment in Japan in 2017. Although this drug is useful to a certain degree, it has a low rate of success (13.3%) (23). According to recent studies, the secondary treatments paclitaxel-cetuximab and paclitaxel-carboplatin with cetuximab therapy have success rates of 54% and 40-56%, respectively. Because the toxicities of these drugs are tolerable and they can be administered on an outpatient basis, these drugs show strong promise as treatments for head and neck cancer and for preserving patient quality of life (2, 24). In particular, studies have reported that paclitaxel-cetuximab is effective in some cases where nivolumab is ineffective and they have also reported on its efficacy (25, 26). However, little research has been conducted on the mechanism of the antitumor effect enhancement obtained through the use of paclitaxel-cetuximab, and on resistance to it. Most studies have focused on monolayer cultures of cell lines or quantitative assessments/assessments of the presence versus absence of gene expression in cancer tissue before the administration of anticancer drugs (27-29), but no reports have focused on restoration of drug sensitivity.

CD-DST, which was used in the present study, has a number of advantages. It allows reproducible, long-term in vitro micro-3D culturing. In addition, anticancer drugs can be used in combination therapies at levels reproducing those found in vivo (13-16). For this reason, we focused in this study on sequential changes in the activation and level of gene expression of NF-Embedded ImageB after exposure to anticancer drugs.

NF-Embedded ImageB is a gene located downstream of the phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) pathway, which is one of the EGFR signal transmission pathways. In addition, it has also been reported that NF-Embedded ImageB activation is involved in breast and colon cancer cell lines (30, 31). Cetuximab competes with EGF for binding to EGFR and disrupts the RAS signal pathway as well as the PI3K-AKT pathway downstream, which gives it its anticancer effect (32). It has been reported that EGFR is expressed in approximately 90% of cases of head and neck cancer (7). However, when mutant KRAS is found in the RAS signaling pathway, it is considered to be one of the causes of resistance to cetuximab, in addition to EGFR blockade and the PI3K-AKT pathway (33). It has also been reported that approximately 5% of head and neck cancer cases bear mutated variants (34-36). We previously investigated whether the eight cell lines used in the present study expressed EGFR and had a KRAS variant and found that all lines showed EGFR expression but none had a KRAS variant (5). Despite this, when we applied CD-DST to genetic changes in AKT, PI3KCA and phosphatase and tensin homolog (PTEN) further downstream of EGFR after treatment with paclitaxel-cetuximab, we found that an increase in PTEN expression was strongly related to the enhanced antitumor effect of paclitaxel-cetuximab (5). This enhancement of the antitumor effect was attributed not only to EGFR expression and KRAS genetic mutation but also to sequential changes in PTEN, which is part of the PI3K-AKT pathway. Studies have observed an antitumor effect by suppressing the activity of NF-Embedded ImageB (37, 38). Restoration of sensitivity might be possible by controlling NF-Embedded ImageB, which is related to changes in the PTEN expression level. As previously stated, these studies were conducted on tissues without prior drug exposure (pretreatment). To our knowledge, there have been no studies on the changes in gene expression of NF-Embedded ImageB after exposure to paclitaxel combined with cetuximab.

Therefore, based on whether or not CD-DST shows sensitivity and an antitumor effect, we divided the samples into the following three groups: the paclitaxel-cetuximab high-sensitivity group; the paclitaxel monotherapy/paclitaxel-cetuximab high-sensitivity group; and the paclitaxel-cetuximab low sensitivity group. We then used real-time PCR and western blot to confirm change in NF-Embedded ImageB expression after exposure to antitumor drugs. The results showed that there was a significant increase in NF-Embedded ImageB expression in all cell lines after monotherapy of paclitaxel or cetuximab. However, with paclitaxel-cetuximab therapy, the cell lines with high sensitivity showed a significant decrease in NF-Embedded ImageB mRNA and protein levels, whereas in those with low sensitivity, NF-Embedded ImageB mRNA and protein levels significantly increased. We observed parallel results in our in vivo tests.

Prior studies reported (9-11) that NF-Embedded ImageB activation is involved in the survival and proliferation of cancer cells as well as resistance to chemotherapy, and that increased activity level of NF-Embedded ImageB, an intracellular protein, is involved in and is a cause of cancer cell proliferation and metastasis (6, 38). Our study’s findings that increased gene expression of NF-Embedded ImageB is correlated with resistance to paclitaxel-cetuximab combined therapy are consistent with this. In their study of monolayer cultures of OSCC cell lines, Harada et al. reported that despite an increase in NF-Embedded ImageB following administration of paclitaxel monotherapy, the antitumor effect was enhanced by the administration of paclitaxel-cetuximab, as cetuximab has a relative suppressive effect on NF-Embedded ImageB activity. (6). Even when paclitaxel was administered alone, we found sensitivity in cell lines with low levels of NF-Embedded ImageB expression compared with other with no sensitivity to monotherapy. This suggests that even when paclitaxel monotherapy triggers an increase in the expression of NF-Embedded ImageB, if the increase in expression level is low, the cytocidal action of paclitaxel results in an antitumor effect, whereas in cases with a significant increase in NF-Embedded ImageB expression, resistance to chemotherapy may occur. This finding suggests that regardless of the drug used, suppression of NF-Embedded ImageB expression after drug exposure may play a major role in subsequent drug sensitivity.

As previously stated, we found increased NF-Embedded ImageB mRNA and protein levels in cell lines with low paclitaxel-cetuximab sensitivity, which strongly suggests a relationship between high NF-Embedded ImageB expression after drug exposure and drug resistance. Because the expression and activity of NF-Embedded ImageB increased after paclitaxel-cetuximab therapy, we investigated whether it is possible to achieve an antitumor effect in the three OSCC cell lines (SAT, HSC-4 and KON) that showed no sensitivity to paclitaxel-cetuximab by suppressing NF-Embedded ImageB.

Bortezomib, a proteasome inhibitor used to suppress NF-Embedded ImageB, is now approved for the treatment of multiple myeloma and mantle-cell lymphoma and is used in a wide variety of other clinical situations (39, 40). Proteasome inhibitors induce cell apoptosis by inhibiting the activity of the NF-Embedded ImageB transcription factor and suppressing the breakdown of the tumor suppressor protein TP53; as a result, proteasome inhibitors are extremely useful due to their antitumor effects (41, 42). Nevertheless, severe adverse drug effects exist, such as peripheral neuropathy, hepatic dysfunction and myelosuppression (43, 44), and caution must therefore be exercised regarding the dose administered and how these drugs are used. The incidence of adverse reactions to bortezomib has been reported to increase in a dose-dependent manner (44, 45). To confirm the antitumor effect of bortezomib on OSCC cell lines using CD-DST, we treated cell lines with the drug at five concentrations. The results showed a dose-dependent antitumor effect, even when used on OSCC cell lines. When 0.2 μg/ml of bortezomib was administered (20), which is the same as the blood concentration of the case treated with multiple myeloma, the cell line with low paclitaxel-cetuximab sensitivity showed high sensitivity. This suggests that bortezomib can be evaluated using CD-DST on OSCC cell lines. In addition, to achieve efficacy while minimizing adverse drug effects, we exposed the same three lines to paclitaxel-cetuximab plus bortezomib at the same five concentrations used for bortezomib monotherapy. We then investigated the antitumor sensitivity of the OSCC cell lines. When paclitaxel-cetuximab with bortezomib was used at a low dose of 0.02 μg/ml, the results indicated significant antitumor efficacy. Although the cell lines that were unresponsive to paclitaxel-cetuximab showed resistance due to high levels of NF-Embedded ImageB expression, the combined use of bortezomib at a low dose suppressed NF-Embedded ImageB, which suggests the possibility of an antitumor effect. Bortezomib monotherapy and combined therapy with other chemotherapy drugs have been found to improve antitumor effects in other solid tumors or OSCC cell lines that express EGFR (46); however, serious adverse drug effects have also been reported (44, 47). The results of the present study suggest the potential of bortezomib in combination with paclitaxel-cetuximab used at a dose of 1/10 of the maximum blood level because antitumor effects can be expected at this dose while minimizing adverse drug effects. In addition, because bortezomib is metabolized by the liver, the International Myeloma Working Group recommends that there is no need to change the dose, regardless of the severity of kidney damage (48). Thus, these results indicate that as a therapy for patients with kidney damage, combining low doses of bortezomib with paclitaxel-cetuximab may result in a further reduction in adverse drug effects, whilst maintaining antitumor efficacy.

Conclusion

The results of this study show that in cases refractory to paclitaxel-cetuximab therapy, the administration of small doses of bortezomib combined with paclitaxel-cetuximab therapy can restore drug sensitivity. However, more research into the doses used in combination therapy is required.

Footnotes

  • Authors’ Contributions

    KT and KS made substantial contributions to the conception and design of the study as well as in the acquisition, analysis and interpretation of the data. KS and AT were involved in the drafting and critical revision of the article for important intellectual content.

  • Conflicts of Interest

    No competing financial interests exist. The Authors have no conflicts of interest to declare.

  • Received June 3, 2022.
  • Revision received June 28, 2022.
  • Accepted July 11, 2022.

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Study of the Acquisition of Sensitivity to Paclitaxel Plus Cetuximab by the Addition of Low-dose Proteasome Inhibitor
MASAKI YODA, KANAME SAKUMA, TOMOYUKI KII, AKIRA TANAKA
Anticancer Research Sep 2022, 42 (9) 4273-4283; DOI: 10.21873/anticanres.15927
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