Abstract

   Medullary thyroid carcinoma (MTC) is a well-known neuroendocrine
   carcinoma, derived from C cells of the thyroid gland. Additionally, MTC
   is an uncommon aggressive carcinoma that metastasizes to lymph nodes,
   bones, lungs and liver. For MTC, the 10-year general survival ratio of
   patients with localized disease is about 95%, whereas that of patients
   with local phase disorder is around 75%. Only 20% of patients with
   distant metastasis to lung at diagnosis survive 10 years, which is
   notably lower than survival for well-differentiated thyroid carcinoma
   (WDTC). The management of MTC with distant metastasis to lung could be
   re-surgery or chemotherapy. In this research, we planned to assess the
   in vitro and in vivo combinational anticancer effect of a novel
   combination of low-dose cisplatin and sorafenib in patient-derived MTC.
   The patient-derived MTC cell lines YUMC-M1, M2, and M3 were isolated
   and treated with a combination of cisplatin and sorafenib or either
   agent alone. Cisplatin and sorafenib acted in combination to forward
   tumor restraint compared with each agent administered alone at a low
   dose. Therefore, a combination of cisplatin and sorafenib could be a
   new therapeutic approach for MTC.

   Keywords: patient-derived medullary thyroid carcinoma, cisplatin,
   sorafenib, cytochrome c, apoptosis

1. Introduction

   Thyroid carcinoma (TC), which accounts for more than 90% of total
   endocrine carcinomas, is the most common endocrine malignancy, and its
   incidence has increased over the last four decades [[32]1]. TC is
   categorized into four main subtypes: papillary TC (PTC), follicular TC
   (FTC), medullary TC (MTC), and anaplastic TC (ATC) [[33]2,[34]3,[35]4].
   MTC progresses from C cells in the thyroid gland and is more aggressive
   and less differentiated than PTC or FTC [[36]5]. MTC occurring from
   parafollicular cells is less typical than WDTC, making up between 2
   [[37]6] and 5% [[38]7] of all thyroid malignancies. Among MTC patients,
   13–15% experience distant metastasis, and the 10-year survival ratio
   was generally 20% [[39]8,[40]9]. Contrasted with differentiated thyroid
   carcinoma (DTC), where the iodine avidity of follicular cells makes
   even metastatic DTC compliant to therapy, parafollicular cells do not
   converge to iodine. Control options for recurrent or metastatic cases
   were re-surgery, radiotherapy and chemotherapy [[41]10]. In addition,
   MTC is less common but encompasses aggressive malignancies with a
   short-term survival and frequent drug-resistance against anticancer
   drugs [[42]11]. The fibroblast growth factor receptor (FGFR) signaling
   pathway is substantially induced in MTC, with the transforming growth
   factor β-mediated epithelial–mesenchymal transition (EMT) contributing
   to poor prognosis [[43]12,[44]13,[45]14]. MTC usually has a good
   prognosis and positive response to therapy, whereas the presence of
   several metastatic MTC indicates a poor prognosis through early
   metastasis and recurrence, and these results can lead to patient death.
   The mission to improve the survival ratio and quality of life of
   patients with untreatable metastatic MTC has produced noteworthy
   studies on other therapies. The progress of targeted therapy has
   brought about major benefits in the control of such patients. Here, two
   drugs, cisplatin and sorafenib, have been approved for use in advanced
   or metastatic MTC [[46]15,[47]16]. According to the report of the NCI
   (National Cancer Institute), sorafenib is a multi-kinase inhibitor
   available for promoting apoptosis and suppressing tumor cell
   proliferation. This is a therapy for a certain type of thyroid cancer
   that targets inhibition of cancer cell growth and new blood vessels
   [[48]17]. Furthermore, cisplatin is used to treat several types of
   cancer and is usually involved as a cytotoxic drug, increasing cancer
   cell death through injuring DNA and preventing DNA synthesis [[49]18].
   Although, the advance of cisplatin- and sorafenib-resistance in
   metastatic MTC is a crucial factor behind the breakdown of conventional
   cancer treatment.

   In this study, we aimed to investigate whether aggressive MTC can be
   suppressed via the induction of cytochrome c-mediated apoptosis in
   response to the combinational anticancer treatment of cisplatin and
   sorafenib at a low dose compared to each agent administered alone.
   These findings could be clinically noticeable for the growth of novel
   combinatorial strategies effectively targeting highly malignant cells
   such as metastatic MTC. In particular, based on these findings, we are
   investigating the fundamental mechanism of anticancer drug resistance
   to forward the expansion of new pharmaceutical composition to address
   this problem.

2. Materials and Methods

2.1. Study Design and Ethical Considerations

   This study was a retrospective, single-center analysis of patients
   diagnosed with MTC (between January 2003 and December 2019), as
   detailed in our previous study [[50]19]. All procedures involving
   patients were performed in accordance with the institutional ethical
   standards, all applicable local/national regulations, and the
   guidelines of the 1964 Helsinki Declaration and its later amendments.
   In accordance with the Bioethics and Safety Act of Korea, formal
   written consent was not required for this type of retrospective,
   observational analysis. The study protocol was approved by the
   Institutional Review Board (IRB) of Severance Hospital, Yonsei
   University College of Medicine (IRB protocol: 3-2019-0281). Cell
   samples were obtained from patients at the Severance Hospital, Yonsei
   University College of Medicine, Seoul, Korea.

2.2. Patients

   Patients who were diagnosed with MTC were pathologically confirmed
   either through surgery or open/core needle biopsy. Patients were
   followed up for at least 1–5 years or until death.

2.3. Patient Tissue Specimens and Clinical Course

   Fresh tumor tissues were collected from patients with histologically
   and biochemical proven MTC who were treated at the Severance Hospital,
   Yonsei University College of Medicine, Seoul, Korea. Fresh tumors were
   collected throughout surgical excision of MTC metastatic and primary
   sites.

   YUMC-M1 was a 24-year-old woman with sporadic MTC (stage II). The
   patient underwent bilateral total thyroidectomy with central
   compartment neck dissection and bilateral modified radical neck
   dissection and has been followed up for 71 months without recurrence or
   metastasis.

   YUMC-M2 was a 19-year-old female MEN2B MTC (stage IVa) patient with
   RET-gene mutation. The patient underwent bilateral total thyroidectomy
   with bilateral modified radical neck dissection and later confirmed her
   pheochromocytoma and underwent left adrenalectomy. The patient has no
   metastasis to other organs, but she has been followed for 76 months
   without her serum calcitonin normalization.

   YUMC-M3 was a 57-year-old man with sporadic MTC with lung, mediastinal
   and axillary lymph nodes metastases (stage IVc). The patient underwent
   bilateral total thyroidectomy with bilateral modified radical neck
   dissection, mediastinal dissection via partial sternotomy and axillary
   lymph node dissection. Since then, metastasis to the axillary lymph
   nodes, retropharyngeal space, and lung has progressed, and he was
   treated with a tyrosine kinase inhibitor, vandetanib.

2.4. Tumor Cell Isolation and Primary Culture

   The patient-derived cancer cells were obtained from fresh tumors of
   patients. YUMC-M1, M2 and -M3 and -M1 were obtained from medullary
   thyroid cancer patients treated at the Severance Hospital, Yonsei
   University College of Medicine, Seoul, Korea. These patients were
   diagnosed with medullary thyroid cancer through fine-needle biopsy
   before surgery and histologically confirmed after surgery. Specimens
   were maintained and transferred to the laboratory. Fat and non-tumor
   parts were eliminated and washed using 1× Hank’s Balanced Salt
   Solution. Cell viability was evaluated with the trypan blue dye.
   Isolated tumor cells were authenticated with short tandem repeat
   profiling, karyotyping, and isoenzyme analysis. Mycoplasmal
   contamination was checked for with the Lookout Mycoplasma PCR detection
   kit (Sigma-Aldrich, St. Louis, MO, USA; MP0035). Further protocol
   details are as described in our previous article [[51]19].

2.5. mRNA-Seq Data

   We pre-processed the raw values from the sequencer to discard low
   attribute and adapter sequences before analysis and revised the
   procedure reads to Homo sapiens (GRCh37) using HISAT v2.1.0. The
   detailed protocol can be found in our previous article [[52]19].

2.6. Statistical Analysis of Gene Expression Level

   The comparative abundances of genes were calculated as read counts
   through StringTie. We carried out the statistical analysis to discover
   differentially expressed genes with evaluations of expression for each
   gene in the samples. The detailed protocol can be found in our previous
   article [[53]19].

2.7. Hierarchical Clustering

   Hierarchical clustering was carried out with complete connect and
   Euclidean distance as a determination of resemblance to show the
   expression patterns of separately shown transcripts, which are
   gratified with |fold change| ≥ 2 and separate t-test raw p < 0.05. The
   whole data analysis was carried out with R 3.5.1 (Vienna, Austria)
   ([54]www.r-project.org, accessed on 1 October 2021).

2.8. Protein–Protein Interaction

   Protein–protein interaction (PPI) was performed through the String
   database ([55]https://string-db.org/, accessed on 20 October 2021) and
   visualized with Cytoscape 3.8.2 ([56]https://cytoscape.org/, accessed
   on 20 October 2021). PPI was classified by a combination score
   (combination scores ≥ 0.4 were associated with the threshold value) and
   connecting whole genes counts (>3 counts).

2.9. Cell Culture

   YUMC-M1, M2, and M3, MTC cell lines were obtained from patient
   specimens. These cells were cultured in RPMI-1640 medium including 15%
   fetal bovine serum (FBS; authenticated by short tandem repeat
   profiling/karyotyping/isoenzyme analysis). The detailed protocol can be
   found in our previous article [[57]19].

2.10. Cell Viability Assay

   Cell viability was estimated by the MTT
   (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium Bromide) assay.
   Tumor cells were cultured in 96-well plates at 7 × 10^3 cells per well.
   These cells were cultured overnight to accomplish 70–80% confluency.
   The indicated drugs were added to achieve final concentrations of 0–40
   μM. The detailed protocol can be found in our previous article
   [[58]19].

2.11. Cell Cycle Analysis Using Flow Cytometry

   Cells were treated with a combination of cisplatin and sorafenib or
   either agent alone in RPMI-1640 medium containing 15% FBS for 40 h. The
   cells were then harvested by trypsinization and fixed in 70% ethanol.
   Further protocol details can be found in our previous article [[59]19].

2.12. Immunofluorescence Analysis and Confocal Imaging

   The level of cytochrome c was evaluated by the immunofluorescence
   staining assay. The primary antibody for the assay was anti-cytochrome
   c (1:25; Abcam, Cambridge, UK) incubated in 3% bovine serum albumin in
   PBS. Nuclei were stained with Hoechst 33342 (Life Technologies, Grand
   Island, NY, USA). Images for immunofluorescence were acquired using a
   confocal microscope (LSM Meta 700; Zeiss, Oberkochen, Germany) and were
   analyzed with Zeiss LSM Image Browser, version 4.2.0121.

2.13. Cellular Fractionation

   Cellular fractionation was performed with the NEPER Nuclear and
   Cytoplasmic Extraction kit (Thermo Scientific, 78833, Waltham, MA, USA)
   in conformity with the manufacturer’s instructions.

2.14. Immunoblot Analysis

   Primary antibodies against CHOP (C/EBP homologous protein, marker of ER
   stress) purchased from Cell Signaling (Cell Signaling Technology,
   Danvers, MA, USA); Bcl-2, cytochrome c, and histone H2B purchased from
   Abcam (Abcam, Cambridge, UK); and caspase 3 and β-actin purchased from
   Santa Cruz (Santa Cruz Biotechnology, CA, USA) were maintained
   overnight at 4 °C. Blots were developed with ECL reagents (Pierce) and
   exposed using Kodak X-OMAT AR Film (Eastman Kodak, Rochester, NY, USA)
   for 3–5 min.

2.15. Human MTC Cell Xenograft

   All experiments were approved by the Animal Experiment Committee of
   Yonsei University. YUMC-M1, M2, and M3 patient-derived MTC cells (5.4 ×
   10^6 cells/mouse) were cultured in vitro and then injected
   subcutaneously into the upper left flank region of female NOD/Shi-scid,
   IL-2Rγ KOJic (NOG) mice. After 15 days, tumor-bearing mice were grouped
   randomly (n = 10 per group) and treated with 5 mg/kg cisplatin (p.o.)
   alone, 80 mg/kg sorafenib (p.o.) alone, and 2.5 mg/kg cisplatin and 40
   mg/kg sorafenib in combination. Tumor size was measured every three
   days using calipers. The detailed protocol can be found in our previous
   article [[60]19]. Whole experiments were confirmed by the Animal
   Experiment Committee of Yonsei University.

2.16. Statistical Analysis

   For statistical analysis of patient information, categorical changes
   were indicated as frequency and proportion, while summary statistics
   (median, range) were used to report consecutive results. Survival
   curves were produced through the Kaplan–Meier method established on the
   log-rank test. The detailed protocol can be found in our previous
   article [[61]19].

3. Results

3.1. Patient Disease Characteristics

   From January 2003 to December 2019, 143 patients were operated on for
   medullary thyroid cancer at Severance Hospital, Yonsei University
   College of Medicine, Seoul, Korea. Patient demographics and disease
   characteristics are presented in [62]Figure 1A. The mean age of the
   patients was 48.9 years, and 62.2% of patients were females. The
   primary tumor diameter was 1.6 ± 1.5 cm, and tumor multiplicity was
   found in 26.6% of total patients. Positive nodal status was observed in
   43.3% of patients, and RET mutation was confirmed in 25% patients.
   Median preoperative serum calcitonin and carcinoembryonic antigen
   levels were 151.5 pg/dL (range: 1.5–8030.0 pg/dL) and 10.0 ng/mL
   (range: 0.8–642.7 ng/mL), respectively. Serum calcitonin normalization
   (upper normal limit of 10 pg/mL) was found in 67.1% patients with MTC.
   Persistent and recurrent lesions were identified in 49 patients; of
   these patients, 32 underwent surgery, 3 received external radiation
   therapy, and 7 received chemotherapy. Of the patients who received
   chemotherapy, four received vandetanib, one received sorafenib and
   adriamycin, one received cabozantinib, and one received epirubicin and
   carboplatin. The median overall survival for 143 patients with MTC was
   72.4 months (range: 4.2–215.5 months; [63]Figure 1A). The patient
   survival rate was 90.7% at 5 years and 79.7% at 10 years after
   detection of TC ([64]Figure 1B).

Figure 1.

   [65]Figure 1
   [66]Open in a new tab

   Information about patients treated for MTC in Severance Hospital: (A)
   patient characteristics and clinical features, and (B) overall survival
   rate of patients with MTC. MTC, medullary thyroid carcinoma.

3.2. Features of Patient-Derived MTC Cell Lines

   Varied MTC cell lines were isolated from the patient specimens
   ([67]Figure 2A); YUMC-M1, M2, and M3 (first, second, and third isolated
   patient-derived MTC) were isolated from patients with MTC treated at
   the Severance Hospital, Yonsei University College of Medicine, Seoul,
   Korea. YUMC-M1, M2, and M3 were more aggressive than FTC, and
   metastasis was confirmed in these patients ([68]Figure 2A). mRNA
   sequencing based on transcriptome analysis to identify a series of
   diversely expressed genes revealed that YUMC-M1, M2, and M3 cells were
   associated with a significant growth of the FGFR signaling pathway and
   EMT markers (ZEB (Zinc finger E-box-binding homeobox), SNAIL (Zinc
   finger protein SNAI1), and TWIST (twist family bHLH transcription
   factor)) were compared with YUMC-F1 (first isolated patient-derived
   FTC), as shown in [69]Figure 2B. In MTC, the most remarkably raised
   genes were FGF (Fibroblast growth factor), FGFR, ZEB, SNAIL, and TWIST
   (twist family bHLH transcription factor) ([70]Figure 2B). Kyoto
   Encyclopedia of Genes and Genomes pathway enrichment analysis
   demonstrated the FGFR signaling pathway and EMT interaction ([71]Figure
   2C).

Figure 2.

   [72]Figure 2
   [73]Open in a new tab

   Characteristics of all examined MTC cell lines: (A) Characteristics of
   patient-derived subtypes of MTC cell lines. (B) Hierarchical clustering
   of annotated genes revealing distinct gene expression. Gene expression
   profiles of patient–derived MTC cells. (C) KEGG pathways connecting to
   the identified proteins reported in STRING database and connecting to
   MTC are highlighted (color coded included in the figure).
   Protein–protein interactions were regenerated based on both known and
   predicted interactions in MTC with STRING analysis. MTC, medullary
   thyroid carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes.

   Taken together, the findings regarding MTC can be of outstanding value
   to therapeutic trials and the administration of patients with
   aggressive TC.

3.3. Combination of Cisplatin and Sorafenib Was More Efficacious Than Either
Agent Alone

   For evaluation of the combinational anticancer efficacy of cisplatin
   and sorafenib on patient-derived MTC cells, we investigated the
   proliferation of YUMC-M1, M2, and M3 cells in either agent combined or
   alone. Cell viability was restrained more efficaciously with a
   combination of cisplatin and sorafenib than with either agent alone in
   a dose-dependent manner ([74]Figure 3A). Notably, the co-treatment had
   a lower half-maximal inhibitory concentration (IC[50]) than that with
   cisplatin or sorafenib treatment alone in YUMC-M1, M2, and M3 cells
   ([75]Figure 3B). These results showed that co-treatment may offer a new
   clinical approach for targeting aggressive MTC with low doses of
   existing anticancer drugs.

Figure 3.

   [76]Figure 3
   [77]Open in a new tab

   Combinational anticancer effects of cisplatin and sorafenib in
   patient-derived MTC YUMC-M1, M2, and M3 cells compared to the effects
   of each agent alone. (A) Cell viability with cisplatin and sorafenib
   combined or each agent alone. All experiments were repeated at least
   thrice. Data represent mean ± standard deviation. * p < 0.05 and ** p <
   0.01 versus control. (B) IC[50] values for the combination of cisplatin
   and sorafenib in patient-derived MTC, YUMC-M1, M2, and M3 cells. Each
   data point signifies the mean of three independent MTT assays,
   performed in triplicate. SEM, standard error of the mean; MTT,
   3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; IC[50],
   half-maximal inhibitory concentration. The asterisk indicates lowest
   half-maximal inhibitory concentration.

3.4. Cisplatin and Sorafenib Co-Treatment Increased Nuclear
Translocation-Mediated Apoptosis in YUMC-M1, M2, and M3 Cells

   Next, we investigated the mechanism of the combinational anticancer
   effects of the co-treatment with cisplatin and sorafenib in YUMC-M1,
   M2, and M3 cells. We analyzed the expression levels of endoplasmic
   reticulum (ER) stress markers (C/EBP homologous protein (CHOP)) and
   apoptotic signaling pathway markers (B-cell lymphoma 2 (Bcl-2) and
   cleaved caspase 3) in YUMC-M1, M2, and M3 cells using flow cytometry,
   immunoblotting (whole-cell lysate or cellular fractionation), and
   immunofluorescence analyses. The combination of cisplatin and sorafenib
   at low doses significantly induced the sub-G[0]/G[1] population,
   resulting in the induction of ER stress-mediated apoptosis ([78]Figure
   4A–C, right panel) in YUMC-M1, M2, and M3 cells compared with either
   agent alone ([79]Figure 4A–C, left panel) and a strong inhibition of
   YUMC-M1, M2, and M3 cells. Immunoblot analysis of the protein
   expression levels indicated that the co-treatment with cisplatin and
   sorafenib led to a marked induction of CHOP and cleaved caspase 3
   levels, which are active apoptosis markers associated with ER stress,
   whereas compared with either agent alone, the co-treatment at low doses
   resulted in a decrease in Bcl-2 level, which is a positive regulator of
   anti-apoptosis. Immunofluorescence analysis showed that cytochrome c
   was translocated to the nucleus, suggesting that the co-treatment with
   cisplatin and sorafenib increased apoptosis via a cytochrome
   c-dependent pathway ([80]Figure 4D). Immunoblot analysis after cellular
   fractionation confirmed that cytochrome c was translocated to the
   nucleus after the co-treatment or treatment with either agent alone
   ([81]Figure 4E). To sum up, these results showed that apoptosis was
   most caused by co-treatment with cisplatin and sorafenib through
   caspase- and cytochrome c-dependent pathways in patient-derived MTC
   cell lines.

Figure 4.

   [82]Figure 4

   [83]Figure 4
   [84]Open in a new tab

   Apoptosis analysis by quantitation of DNA content with propidium iodide
   and immunoblot analysis. (A–C) Flow cytometry (left panel) and
   immunoblot analysis (right panel) of YUMC-M1, M2, and M3 cells. Cells
   were exposed to the indicated inhibitors, harvested, and stained with
   propidium iodide before analysis by flow cytometry and FlowJo version
   8, Ashland, Dickinson and Company; 2021. Immunoblot analysis of the
   markers of endoplasmic reticulum stress, apoptosis, and anti-apoptosis
   in patient-derived MTC cell lines, YUMC-M1, M2, and M3. (D,E) Cisplatin
   and sorafenib highly induced nuclear translocation of cytochrome
   c-mediated apoptosis in patient-derived MTC cell lines, YUMC-M1, M2,
   and M3. (D) Immunofluorescence examined at 40× magnification; scale
   bar: 20 μm; and (E) subcellular fractionation analysis. Cytochrome c
   was most translocated and accumulated in the nucleus in the cisplatin
   and sorafenib co-treatment group.

3.5. Cisplatin and Sorafenib Co-Treatment Remarkably Restrained Tumor Growth
in Mouse Xenograft Model with Patient-Derived MTC Cell Lines

   To assessment the combinational in vivo anticancer effectiveness of the
   co-treatment with cisplatin and sorafenib, we developed a mouse
   xenograft model with YUMC-M1, M2, and M3 patient-derived MTC cell lines
   ([85]Figure 5). We discovered that cisplatin or sorafenib alone could
   restrain tumor growth, but compared with either agent alone, the
   co-treatment clearly increased tumor shrinkage at low doses ([86]Figure
   5A,D,G). Appreciably, YUMC-M3 was not markedly impacted by cisplatin or
   sorafenib alone. Moreover, mice in the cisplatin and sorafenib
   co-treatment group had significantly smaller excised tumor weight than
   that of mice administered with cisplatin or sorafenib alone ([87]Figure
   5B,E,H). There was no proof of systematic toxicity and death in any
   group. The body weight of mice did not remarkably differ among all
   groups ([88]Figure 5C,F,I).

Figure 5.

   [89]Figure 5
   [90]Open in a new tab

   Anticancer effects of cisplatin and sorafenib in combination or with
   each agent alone in patient-derived MTC cell lines, YUMC-M1, M2, and M3
   cell xenografts in vivo. (A,D,G) Change in tumor volume. (B,E,H) The
   dissected tumor weight. (C,F,I) Change in whole body weight. Cisplatin
   and sorafenib had no significant effect on mouse body weight. * p <
   0.05 and ** p < 0.01, compared with control.

   Taken together, the combination of cisplatin and sorafenib indicated
   potent anticancer effects in an aggressive patient-derived MTC cell
   xenograft model.

4. Discussion

   The survival ratio of thyroid carcinoma is well-known as being the best
   among general endocrine-mediated malignancies [[91]20], and its onset
   rate is consistently growing worldwide, as well as specifically in the
   Korea [[92]20,[93]21,[94]22,[95]23]. Thyroid carcinoma is poised to
   become the fourth leading carcinoma worldwide [[96]22]. For the past 20
   years, the global age-standardized incident ratio of thyroid carcinoma
   has raised by roughly 25% [[97]22,[98]24,[99]25]. This worldwide
   expansion has been ascribed to, e.g., raised factors, early disclosure
   of cancer, induced environmental hazard elements, and iodine degrees
   [[100]22]. MTC is known to be an uncommon endocrine carcinoma that
   occurs in thyroid parafollicular C cells [[101]26,[102]27]. In
   particular, various investigations have shown that advanced or
   metastatic MTC has demonstrated refractory to almost medical therapies
   [[103]28,[104]29]. Metastasis of the MTC was via blood and lymphatic
   vessels, which had more aggressive clinical behavior than DTC.
   Rearranged during Transfection (RET) gene mutation is known as a
   central molecular change in MTC [[105]30,[106]31,[107]32]. In germline
   RET mutation, MTC is classified under three different phenotypes:
   familial MTC, and multiple endocrine neoplasia types 2A and 2B (MEN2)
   [[108]33,[109]34]. Moreover, somatic RET mutation stands as the major
   molecular change in almost all cases [[110]35,[111]36,[112]37]. In
   surgical cases, patients could choose whole thyroidectomy with
   prophylactic or remedial pivotal section lymph node resection
   [[113]38]. However, unfortunately, in the case of large metastasis,
   especially in those with remarkably induced tumor growth, extra
   remedies were required. Moreover, systemic treatment of advanced or
   metastatic MTC is limited at present to administration of a tyrosine
   kinase inhibitor (TKI) [[114]39,[115]40,[116]41,[117]42]. Here, we
   report the combinational anticancer efficacy of cisplatin and sorafenib
   at a low dose compared with each treatment agent alone in
   patient-derived advanced MTC. Cytochrome c release from mitochondria is
   a critical event for apoptotic death [[118]43,[119]44]. Distinctly,
   cytochrome c release was significantly induced in the caspase-dependent
   apoptosis signaling pathway [[120]45,[121]46]. We indicted that release
   of cytochrome c from mitochondria most significantly increased
   apoptosis, and progressively amassed in the nucleus as proven by both
   immunofluorescence and cellular fractionation after medication of
   cisplatin and sorafenib at a low dose. Clinically aggressive MTCs lead
   to poor prognosis [[122]47], considered as refractory carcinoma
   subtypes owing to their aggressive behavior [[123]48]. Accordingly,
   there is an imminent need to investigate new clinical approaches for
   the remedy of aggressive MTCs.

   In this research, we showed a prospective new therapy scheme for
   advanced MTC ([124]Figure 6). Of course, our proposal might be the tip
   of the iceberg to numerous clinical approaches for therapy of advanced
   MTC. In particular, the study of patient-derived MTC was difficult and
   limited. Nevertheless, if these challenges continue to pile up, there
   could be a breakthrough for a clinical solution of advanced MTC.
   Clinically, these findings convey critical implications for the
   progress of novel combinatorial strategies that target selective
   drug-resistant cancer cells.

Figure 6.

   [125]Figure 6
   [126]Open in a new tab

   Scheme of study design.

5. Conclusions

   According to the guidelines of the National Cancer Institute, cisplatin
   and sorafenib are already well-known anticancer drugs. Although, in
   conventional cancer therapy, there is no definite solution for
   treatment of recurrent or metastasis by refractory MTC. In these
   research findings, the combinational anticancer consequences of the
   cisplatin and sorafenib were more capable at low doses of each agent
   alone against patient-derived metastatic MTC. These results could be
   practical in the design of prospective therapeutic trials and aid the
   expansion of valuable therapies for patients with refractory MTC.
   However, research on patient-derived refractory cancer cells is
   challenging and limited. However, these challenges could be cause to a
   breakthrough in, and clinical solution to, refractory cancer therapy.

Acknowledgments