Hinokitiol Dysregulates Metabolism of Carcinoma Cell Lines and Induces Downregulation of HPV16E6 and E7 Oncogenes and p21 Upregulation in HPV Positive Cell Lines

Background: Hinokitiol (beta-thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological properties including anti-inflammatory, anti-microbial, and anti-tumor effects. Therefore, hinokitiol has become a frequent additive in oral and other healthcare products. Objectives: Our goal was to determine the anti-tumor activity of hinokitiol on human papillomavirus (HPV) positive (n = 3) and negative (n = 2) cell lines derived from cervical or head and neck squamous cell carcinoma (HNSCC) and keratinocyte cell lines (n = 3) transformed spontaneously or with HPV16E6 and E7 oncogenes. Methods: The cell-lines were exposed to hinokitiol at different concentrations (0-200 mu M) for 24 h. Cell metabolism, proliferation, and the cell cycle distribution were assessed by MTT- and H-3-thymidine incorporation and flow cytometry. Expressions of p21 and on HPV16E6 and E7 oncogenes were assessed by qPCR. Results: In all carcinoma cell lines, hinokitiol treatment declined the metabolic activity irrespective of the HPV status. This decline was statistically significant, however, only in HPV-positive cell lines CaSki and UD-SCC-2 when exposed to hinokitiol concentrations at 100 and 200 mu M, respectively (p Conclusions: Our results indicate that hinokitiol might have potential in preventing the progress of immortalized cells toward malignancy and the growth of malignant lesions. Hinokitiol can also influence on the progression of HPV-associated lesions by downregulating the E6 and E7 expression.</p

Hinokitiol Dysregulates Metabolism of Carcinoma Cell Lines and Induces Downregulation of HPV16E6 and E7 Oncogenes and p21 Upregulation in HPV Positive Cell Lines

Background: Hinokitiol (β‐thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological properties including anti‐inflammatory, anti‐microbial, and anti‐tumor effects. Therefore, hinokitiol has become a frequent additive in oral and other healthcare products. Objectives: Our goal was to determine the anti‐tumor activity of hinokitiol on human papillomavirus (HPV) positive (n = 3) and negative (n = 2) cell lines derived from cervical or head and neck squamous cell carcinoma (HNSCC) and keratinocyte cell lines (n = 3) transformed spon-taneously or with HPV16E6 and E7 oncogenes. Methods: The cell‐lines were exposed to hinokitiol at different concentrations (0–200μM) for 24 h. Cell metabolism, proliferation, and the cell cycle distribution were assessed by MTT‐ and3H‐thymidine incorporation and flow cytometry. Expres-sions of p21 and on HPV16E6 and E7 oncogenes were assessed by qPCR. Results: In all carcinoma cell lines, hinokitiol treatment declined the metabolic activity irrespective of the HPV status. This decline was statistically significant, however, only in HPV‐positive cell lines CaSki and UD‐SCC‐2 when exposed to hinokitiol concentrations at 100 and 200 μM, respectively (p < 0.05). Immortalized cell lines, HMK and HPV‐positive IHGK, were more sensitive as a similar metabolic effect was achieved at lower hinokitiol concentrations of 3.1, 6.25, and 50 μM, respectively. Hinokitiol blocked DNA synthesis of all carcinoma cell lines without evident association with HPV status. G1 cell cycle arrest and p21 upregulation was found in all cell lines after hinokitiol treatment at higher concen-tration. However, when the p21 results of all HPV‐positive cells were pooled together, the increase in p21 expression was statistically significantly higher in HPV‐positive than in HPV‐negative cell lines (p = 0.03), but only at the highest hinokitiol concentration (200 μM). In HPV‐positive cell lines hinokitiol declined the expression of HPV16E7 and E6 along the increase of p21 expression. The dose‐dependent inverse correlation between p21 and E7 was statistically significant in SiHa cells (r = −0.975, p‐value = 0.03) and borderline in UD‐SCC‐2 cells (r = −0.944, p‐value = 0.06), in which p21 and E6 were also inversely correlated (r = −0.989). Conclusions: Our results indicate that hinokitiol might have potential in preventing the progress of immortalized cells toward malignancy and the growth of malignant lesions. Hinokitiol can also influence on the progression of HPV‐associated lesions by downregulating the E6 and E7 expression.publishedVersionPeer reviewe

HINOKITIOL IMPAIRS CELL VIABILITY AND PROLIFERATION OF HUMAN PAPILLOMAVIRUS (HPV)-IMMORTALIZED AND CARCINOMA CELL LINES

Hinokitiol (β-Thujaplicin), isolated from the wood of Chamaecyparis taiwanensis, has a wide variety of biological activities including anti-inflammatory, anti-microbial and anti-tumour effects. Because of these activities, hinokitiol has been widely used in oral and other health care products. It also has been recognized to have significant anti-microbial and cytotoxic activities against oral pathogens and oral squamous cell carcinoma cell lines. Because of its multiple biological activities, hinokitiol might display a high potential for safe and effective applications in oral health care. In this study our goal was to determine the cytotoxic and anti-tumour activity of hinokitiol on immortalized and carcinoma cell lines as related to the status of human papillomavirus (HPV) infection. We treated nine cell lines (four HPV16-positive and five HPV-negative) with hinokitiol at different concentrations (0-200 µM) for 24 hours. Of these cell lines, three were established from HPV16 positive and two from HPV-negative squamous cell carcinomas. Also, three immortalized cell lines (transformed either spontaneously or with HPV16) and oral fibroblasts were tested. Cell viability and proliferation were assessed by MTT- and 3H thymidine incorporation assays. Hinokitiol (50-200 µM) did suppress cell viability of immortalized and carcinoma cell lines without evident association with HPV status. The cytotoxic effect of hinokitiol on transformed cell lines differed from that found in carcinoma cell lines. Hinokitiol at the low concentration (6.25 µM) was able to suppress cell viability of immortalized cells up to 80% in HPV-negative HMK but also to lesser degree in HPV16-positive immortalized IHGK cells. In the 3H thymidine incorporation assays, hinokitiol suppressed DNA synthesis in all cell lines and the effect was similar in both HPV-positive and -negative cell lines. In fibroblasts, hinokitiol had only a minor effect at higher concentrations. Our finding that hinokitiol is more toxic to immortalized cell lines than to carcinoma cell lines, indicates that hinokitiol might prevent immortalized cells to progress toward malignancy. In this study, no evident differences in hinokitiol effect on HPV-positive and -negative cell lines were found. However, detailed analyses and more studies are urgently needed to further understand the antitumour effects of hinokitiol on immortalized cell lines and HPV.Hinokitioli (β-thujaplisiini) on sypressipuulajista eristetty luonnon yhdiste, ja sillä on havaittu olevan mikrobien kasvua ja lisääntymistä estäviä vaikutuksia. Näiden ominaisuuksiensa vuoksi hinokitiolia on käytetty erilaisissa kosmetiikka- ja suunhoitotuotteissa. Tutkimusten mukaan hinokitiolilla on lisäksi havaittu olevan suurempi sytotoksinen vaikutus suun alueen syöpäsoluja vastaan verrattuna suun normaaleihin keratinosyyttisoluihin. Antimikrobisten ja syöpää ehkäisevien ominaisuuksiensa vuoksi hinokitioli nähdäänkin potentiaalisena suun terveyttä edistävänä aineena. Tämän tutkimuksen tavoitteena oli määrittää hinokitiolin sytotoksisia ja antituumorisia vaikutuksia immortalisoituneisiin sekä syöpäsolulinjoihin suhteessa niiden HPV-infektion statukseen. Tutkimme hinokitiolin vaikutuksia yhdeksään eri solulinjaan, joista neljä oli HPV-positiivisia ja viisi HPV-negatiivisia. Näistä viisi oli levyepiteelikarsinoomista eristettyjä syöpäsolulinjoja ja kolme spontaanisti immortalisoituneita solulinjoja. Lisäksi verrokkina käytettiin suun normaaleja fibroblasteja. Solut altistettiin eri pitoisuuksille (0-200 µM) hinokitiolia 24 tunnin ajaksi. Tämän jälkeen solujen elinkykyä mitattiin MTT-määrityksellä ja jakaantumista 3H-tymidiini-inkorporaatiolla. Hinokitioli (50-200 µM) laski solujen elinkykyä niin immortalisoituneissa kuin syöpäsolulinjoissakin riippumatta niiden HPV-infektion statuksesta. Lisäksi hinokitiolin havaittiin laskevan immortalisoituneiden HMK- ja IHGK-solujen elinkykyä jo matalalla konsentraatiolla (6,25 µM). Myös solujen jakautumista mittaavassa tymidiinitesteissä hinokitiolin vaikutus nähtiin samanlaisena riippumatta solujen HPV-statuksesta, ja hinokitioli vähensi solujen DNA-synteesiä annosriippuvaisesti sekä immortalisoituneissa että syöpäsolulinjoissa. Ihmisen normaaleihin fibroblasteihin hinokitiolin vaikutus oli vähäinen suuremmillakin (200 µM) annoksilla. Näin ollen hinokitiolin käyttö vaikuttaisikin olevan turvallista pienillä pitoisuuksilla. Lisäksi löydöstemme perusteella hinokitioli saattaisi ehkäistä immortalisoituneissa soluissa kehitystä maligniin suuntaan. Jotta hinokitiolin syöpää ehkäiseviä ominaisuuksia voitaisiin ymmärtää paremmin, on jatkotutkimuksille tarvetta. Lisäksi yksityiskohtaisemmat analyysit solutason mekanismeista voisivat osoittaa eroja hinokitiolin vaikutuksesta HPV-positiivisten ja -negatiivisten solujen välillä, vaikka tässä tutkimuksessa eroja ei ollut nähtävissä