Fisetin inhibits laryngeal carcinoma through regulation of AKT/NF-κB/mTOR and ERK1/2 signaling pathways.

Targeting cancer cells is crucial for improving the efficiency of laryngeal cancer treatment. However, the signaling pathway and therapeutic strategy, related to the tumor, still need further research. Dietary flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) found in many fruits and vegetables has been shown in preclinical studies to inhibit cancer growth through regulating cell cycle, apoptosis, angiogenesis, invasion and metastasis without causing any toxicity to normal cells. PI3K/AKT and ERK1/2 have been known as essential signaling pathways to modulate cell proliferation, apoptosis as well as autophagy via mTOR, Caspase-3 and NF-κB signals. In our study, flow cytometry and western blot assays suggested that apoptosis was induced by fisetin administration, promoting Caspase-3 expressions by regulating PI3K/AKT/NF-κB. Additionally, fisetin suppressed TU212 cells proliferation, which was linked with ERK1/2 inactivation. Further, the activation of PI3K/AKT-regulated mTOR was inhibited by fisetin, leading to transcription suppression and proliferation inhibition of TU212 cells. In vivo studies also showed that the tumor volume and weight of nude mice were reduced for fisetin use with KI-67 decrease and LC3II increase in tumor tissue samples. Together, our data indicated that fisetin had a potential role in controlling human laryngeal cancer through inhibiting tumor cell proliferation, inducing apoptosis and autophagy regulated by ERK1/2 and AKT/NF-κB/mTOR signaling pathways, which might provide a therapeutic strategy for laryngeal cancer inhibition in future.

miR-219 inhibits the growth and metastasis of TSCC cells by targeting PRKCI.

Tongue squamous cells carcinoma (TSCC) is the most common type in oral cancers. Recently, accumulating evidence suggests that microRNAs (miRNAs) play critical roles in tumorigenesis. Here, we demonstrated that miR-219 was significantly downregulated in TSCC tissues and cell lines. miR-219 overexpression remarkably suppressed cell proliferation, colony formation, migration and invasion of TSCC cells. In addition, protein kinase CI (PRKCI) was identified as a target of miR-219, and overexpression of PRKCI could significantly attenuated the tumor suppressive effects of miR-219. Furthermore, PRKCI inversely correlates with miR-219 in TSCC tissues. Taken together, miR-219 inhibited growth and metastasis by targeting PRKCI and might be used as a potential target for the treatment of TSCC.

[Construction and confirmation of a recombinant adenovirus vector of survivin].

OBJECTIVE: To construct a recombinant adenovirus of survivin vector and provid valuable reference for gene therapy of laryngeal cancer. METHODS: The survivin gene was cloned by PCR. After confirmation by enzyme restriction analysis and sequencing, the gene and the adenovirus vector were recombined together to construct the recombinant adenovirus vector. The recombinant adenovirus vector was confirmed via both sequencing and digestion restriction analysis, and then linearized and transfected into the HEK 293 cell line to generate recombinant adenovirus. RESULTS: The sequence analysis demonstrated that the survivin gene sequence was the same as published in the literature, suggesting that a recombinant adenovirus vector has been successfully constructed. CONCLUSIONS: A survivin recombinant adenovirus has been successfully constructed.

[Neck dissection of Levels II and III for the treatment of supraglottic laryngeal cancer with clinically negative neck].

OBJECTIVE: To evaluate the feasibility of routine inclusion of levels II and III in neck dissection to treat the occult neck metastasis as elective treatment for supraglottic cancer with clinically node negative (cN0). METHODS: The results of 52 cN0 patients with supraglottic cancer treated in Tumor Hospital, Harbin Medical University from October 2002 to March 2006 were reviewed retrospectively. RESULTS: Of the 52 patients with supraglottic cancer and cN0 neck, 32 cases received ipsilateral SND (levels II and III) and 20 cases with bilateral SND (levels II and III). Fifteen (28.9%) of 52 patients were found to have occult regional metastasis on pathological examination. Three patients without metastasis in dissected side at pathologic examination showed metastasis in the contralateral undissected neck later on therefore the total occult metastasis rate was 34.6% (18 of 52). The unilateral and bilateral neck occult metastases were determined in 15 cases (28.9%) and 3 cases (5.8%) respectively. A total of 1190 lymph nodes were harvested in 72 specimens, with 30 positive nodes. The distributions of the 30 positive nodes were as follows: level IIA 83.3% (25 nodes), level III 16.7% (5 nodes). Three-year regional recurrence rate was 5.8%. The 3-year survival rate was 84.6% according to Kaplan-Meier in all cN0 patients (n = 52). Patients with positive neck metastasis and patients with extracapsular spread had higher regional recurrence rates (P = 0.021 and 0.002, respectively). CONCLUSIONS: The results support the use of SND (levels II and III) in cN0 supraglottic cancer. This procedure will reduce both operating time and morbidity, without compromising the oncologic result.

[Expression of survivin gene in thyroid carcinoma and its significance].

OBJECTIVE: To study the expression of survivin gene in thyroid carcinoma and its significance. METHODS: Samples of thyroid carcinoma were collected from 98 patients during operation. Western blotting and RT-PCR were used to detect mRNA and protein expression of survivin in the carcinoma tissue and the tissue around the carcinoma. TUNEL method was used to detect the apoptotic index (AI). RESULTS: Western blotting and RT-PCR methods showed similar results. The positive expression rates of survivin protein and mRNA in the thyroid carcinoma were both 68.37%; however, survivin was not expressed in all of the tissues around the thyroid carcinoma at both the mRNA and protein levels. The expression rate of survivin was closely related to the pathological typing, lymph node metastasis, and hematogenous metastasis. Apoptotic cells were found in all thyroid carcinoma. The mean AI of the surviving positive group was 0.48 +/- 0.38, significantly lower than that of the survivin negative group (2.98 +/- 0.12, P = 0.0082). CONCLUSION: Survivin gene is expressed in most of the thyroid carcinoma. The positive expression rate of survivin is significantly linked to cell proliferating activity, lymph node metastasis, and hematogenous metastasis. Survivin gene will be a new target of gene therapy in thyroid carcinoma.

[Expression of drug resistance and metastasis related gene in adenoid cystic carcinoma and mucoepidermoid carcinoma].

OBJECTIVE: To examine the differential gene expression among adenoid cystic carcinoma ACC-M, low metastasis ACC-2 and low differentiated mucoepidermoid carcinoma MEC-1 with gene array, and detect three important genes with real-time quantitative PCR. METHODS: Gene array was used to screen the differential genes, then three important genes were selected and detected by real-time quantitative PCR. RESULTS: The gene contents of MDR-1, MRP-1 and CCD1 were MEC-1 > ACC-M > ACC-2, MEC-1 > ACC-M > ACC-2 and MEC-1 > ACC-M > ACC-2 respectively. CONCLUSIONS: Drug resistance of MEC-1 is related with high content of MDR-1. High metastasis of ACC-M is concerned with high content of MRP-1 and low content of CCD-1. Low metastasis of ACC-2 has a relationship with low content of MRP1.

[Supraglottic horizontal partial laryngectomies in 163 cases].

OBJECTIVE: To evaluate the clinical value of supraglottic horizontal partial laryngectomy. METHODS: One hundred and sixty-three patients with supraglottic laryngeal carcinoma were treated surgically by supraglottic horizontal partial laryngectomy from 1978 to 1998. There were 64 males and 99 females. Five cases were staged I, 95 staged II, 48 staged III and 15 staged IV. The surgical techniques were improved: The hyoid was removed conventionally; The outer perichondrium of thyroid cartilage was turned into the laryngeal cavity and sutured with the mucosa of laryngeal ventricle and the base of tongue was sutured to the reserved thyroid cartilage. Survival was evaluated using the Kaplan-Meier method. The differences between stages were tested by Los-Rank method. RESULTS: The 5-year survival rate were 100.0%, 77.9%, 54.2% and 33.3% for patients staged I to IV respectively, and were statistically significant (P = 0.0006) between different clinical stages. The 5-year survival rate were 73.1% and 45.5% in patients with cN0 and cN + respectively, and were statistically significant (P = 0.0132). The speech and swallowing functions were good after operation. The decannulation rate was 91.4%. The main causes of death were cervical lymph node metastasis in 40% (20/50) and laryngeal recurrence in 18% (9/50). The occult metastasis rate was 23.1% (30/130) and cervical metastasis rates of patients with cN1-3 75.8% (25/33), with a total metastasis rate was 33.7 (55/163). CONCLUSIONS: Supraglottic horizontal partial laryngectomy is very effective in eradicating disease and in preserving laryngeal function on condition that the indications were selected correctly. The selective lateral neck dissection was recommended for supraglottic carcinoma.

[Effect of survivin antisense oligodeoxynucleotide on carcinoma of larynx in vivo and in vitro].

OBJECTIVE: To observe the effect of survivin antisense oligodeoxynucleotide (ASODN) on the apoptosis of human carcinoma of larynx cell line Hep2 and the inhibitory rate in nude mice model so as to discuss the selective blocking activity of antisense technique on gene expression seeking a new way for gene therapy of carcinoma of larynx. METHODS: Antisense oligodeoxynucleotides survivin were transformed into human carcinoma of larynx cell line Hep2 by liposome Lipofectamine 2000. Within 72 h after transfection, 3-(4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) assay was used to detect cellular proliferation. Forty eight hours after transfection, reverse transcription-polymerase chain reaction (RT-PCR) assay was used to observe the expression of survivin gene, Western Blot assay for the protein, and terminal deoxynucleotide mediated nick end labeling (TUNEL) and flow cytometer for cellular apoptosis. RESULTS: Cellular inhibition rate of 72 h went up to 52. 5% and 71.4% at 1.0 micromol/L and 2.0 micromol/L value in Lipo-ASODN groups which differed statistically remarkably (P = 0.046), higher than that in controls in MTT assay (P =0. 003 and 0. 0004). Forty eight hours after transfection survivin gene expression in Lipo-ASODN groups were less than that in control group in RT-PCR assay. Survivin protein expression decreased in Western blot. In TUNEL assay, nuclear positive staining was observed and the apoptosis peak was observed in flow cytometer test, which were absent in controls. In nude mice of carcinoma of larynx model, the inhibitory rate in Lipo-ASODN groups got up to 48.1% and 61.3% higher than that of controls (P < 0.004 and 0. 0006), which differed remarkably (P = 0.032) in a dose-dependently way. CONCLUSIONS: The findings showed that the expression of survivin gene and protein induced cellular apoptosis in Hep2 cells after transfection of Lipo-ASODN and that the carcinoma of larynx in the nude mice model were inhibited by Lipo-ASODN which suggested that antisense technique can be an effective means in the gene therapy of carcinoma of larynx.

[An observation on long-term results of subtotal laryngectomy with preservation of an arytenoid cartilage].

OBJECTIVE: To discuss the long-term results of the subtotal laryngectomy with preservation of an arytenoids cartilage. METHODS: The long-term results of the subtotal laryngectomy with preservation of an arytenoids cartilage were analysed in 82 patients with squamous cell carcinoma of larynx (stage III 73 cases, IV 9 cases; supraglottic type 71 cases, glottic 6, transglottic 5). RESULTS: The functions of speech and deglutition were recovered in 82 patients, and the respirations were normal in 69 of 82 cases (84.2%). Pharyngeal fistula appeared in 2 cases, and the infection of incision wound in 6, but all of them were cured by conservative treatment. All of 82 cases were followed-up for more than 5 years, 5 cases were lost follow-up, so the follow-up rate was 93.3%. 26 patients died of various causes during 5 years after operation. 5 of 26 cases died of local recurrence of laryngeal carcinoma, neck metastasis in 4, others in 4 (cardiac insufficiency, myocardiac infarction, lungs metastasis, rectal carcinoma 1 case, respectively) unknown in 4, lost follow-up in 5.68 patients were followed-up for more than 10 years, 30 cases were dead (26 cases who died of various causes included during 5 years after operation; died of local recurrence in larynx was 1 case, unknown 2, lost follow-up 1, during 5 approximately 10 years after surgery). 5, 10 years survival rates were 68.3% (56/82) and 55.9% (38/68), respectively. CONCLUSIONS: Subtotal laryngectomy with preservation of an arytenoids cartilage is recommended for T3 and some elective T4 laryngeal carcinoma.

[Distribution of cervical lymph node metastasis in patients with cN1 supraglottic cancer].

OBJECTIVE: To study the characteristics of the cervical lymph node metastasis of patients with cN1 supraglottic cancer and to elucidate which levels should be involved during neck dissection. METHODS: Modified neck dissections or radical neck dissections were performed for 108 cases (147 sides) of cN1 supraglottic cancer. All the lymph nodes of the samples of 147 operation sides were examined pathologically to identify the metastatic levels. RESULTS: Among samples of 108 cases (147 sides), 126 lymph nodes were metastasis, among which 113 lymph nodes were located in levels II and III (89.7%, 113/126), and 123 lymph nodes in levels II, III and IV (97.6%, 123/126). The 126 metastatic nodes were distributed in the 109 levels and 96 of them were in the levels II and III (88.1%, 96/109) and 106 were in the levels II, III and IV (97.2%, 106/109). Additionally, 45 cases (63 sides) of the 108 patients were pathologically metastasis (41.7%) and bilateral cervical metastasis occurred in 20 cases (18.5%). The cervical recurrent rate was 7.4% (8/108) and occurred in the levels II, III and IV respectively, during five to fourteen years follow-up. CONCLUSION: Levels II, III and IV dissection may be reasonable for patients with cN1 supraglottic carcinomas, levels I and V dissection may be avoidable when pathological evidence of metastasis in levels I and V has not been obtained. Contralateral lymph node dissection at the levels II, III and IV should be performed in the case of ipsilateral pN + or contralateral metastasis cN1.

[Pathological feature and management of occult lymphatic metastasis in supraglottic carcinoma].

OBJECTIVE: To study the pathologic feature and management methods of occult lymphatic metastasis in patients with supraglottic carcinoma. METHODS: The following are criteria of patients selection: (1) Supraglottic squamous cell carcinoma; (2) Clinical N0M0; (3) no preoperative radiotherapy and (or) chemotherapy. Thirty patients were recruited, male 19 cases, female 11; age: 40 - 72yr, average 54.8yr; T2 8 cases, T3 18, T4 4 (by UICC 1997). Ipsilateral supraomohyoid neck dissections were performed in all cases. Lymph nodes were studied histologically according to the levels of dissection. RESULTS: Six of 30 cases were positive nodes histologically in first operation, 3 were occurrence neck metastasis in opposite side during follow ups. Occult metastasis rate was 20% (6/30) ipsilaterdly and 10% contralaterally. 527 lymph nodes were collected in all of 30 patients, average 17.6 nodes in every side neck. Ten positive lymph nodes histologically were harvested. The distribution of metastatic lymph nodes was 9 in level II, 1 in level III, no in level I. N0 recurrence in larynx and (or) at the neck after dissection. Two years survival rates was 86.7% (26/30) without tumor. CONCLUSION: Occult metastasis rate of supraglottic carcinoma is as high as 30%. The selective lateral neck dissection of level II, III and occasionally, level IV was recommended.

[Immune responses against cancers in cervical lymph nodes of laryngeal carcinoma].

OBJECTIVE: Densities of dendritic cells (DC) and hyperplastic follicular response in cervical lymph nodes were performed to prove their roles in immune responses against cancers. METHODS: Paraffin blocks were prepared for staining with monoclonal antibodies against CD45RO, CD20 and S-100 proteins,in 157 lymph nodes obtained from elective cervical lymphadenectomy in 47 cases of laryngeal squamous cell carcinomas. RESULTS: Quantitative analysis showed that the patients who survived longer than 5 years had significant higher number of follicles and higher extent of infiltration by DCs in the lymph nodes than those who less than 5 years (P < 0.001). According to negative or positive lymph node metastasis, there were statistically significant differences between two groups (P < 0.001). The patients who possessed T cell increase type follicular reaction had significant higher five-year survival rate ( P < 0.01) and lower lymph node metastasis rate (P < 0.05) than those who possessed T cell decrease type reaction. CONCLUSION: DCs and hyperplastic follicular response may be more directly involved in the host immune reaction against tumor. The classification of follicular reaction, the densities of DCs and follicular reaction, can serve as important indicators in assessing prognosis of laryngeal carcinomas.

[Cervical metastasis in patients with T2-4 cN0 laryngeal carcinoma].

OBJECTIVE: To study the characteristics of the cervical lymph node metastasis in clinical N0 (cN0) patients with laryngeal carcinoma and its implication in clinical treatment. METHODS: 76 patients with laryngeal carcinomas of T2-4cN0 category were divided into two groups in random: 21(22 sides) radical neck dissection(RND) and 55(60 sides) functional neck dissection(FND) were performed. Lymph nodes were studied histologically according to the levels. RESULTS: On an average, 29.6 lymph nodes were obtained in one side of neck in RND group, and 24.7 in FND group(F = 3.145, P = 0.068). The occult metastasis rates were 33.3% (7/21) in RND group and 34.5% (19/55) in FND group. 25 of 26 patients (96.2%) who had positive nodes involved only the levels II and III. 2130 lymph nodes were obtained in all samples, 59 of 60 positive nodes(98.3%) were located in the level II and III. The 5 and 10-year survival rates of the two groups were 71.4% (15/21), 76.4% (42/55) and 61.9% (13/21), 68.9% (31/45), respectively with no statistical difference(chi 2 = 0.2394, P > 0.5; chi 2 = 0.3143, P > 0.05). Ipsilateral cervical recurrence rates in two groups were 9.5% (2/21) and 7.3% (4/55), respectively with no statistical difference (chi 2 = 0.1059, P > 0.900). 10-year mortalities with negative and positive cervical lymph nodes were 16.7% (7/42) and 62.5% (15/24) respectively, which had statistically difference (chi 2 = 14.4375, P < 0.005). CONCLUSION: The lateral neck (level II, III and IV) dissection may be suitable for the treatment laryngeal carcinoma patients with T2-4cN0.