Tumour eradication using synchronous thermal ablation and Hsp90 chemotherapy with protein engineered triblock biopolymer-geldanamycin conjugates

Purpose: Hepatocellular carcinoma (HCC) suffers high tumour recurrence rate after thermal ablation. Heat shock protein 90 (Hsp90) induced post-ablation is critical for tumour survival and progression. A combination therapy of thermal ablation and polymer conjugated Hsp90 chemotherapy was designed and evaluated for complete tumour eradication of HCC. Materials and methods: A thermo-responsive, elastin-like polypeptide (ELP)-based tri-block biopolymer was developed and conjugated with a potent Hsp90 inhibitor, geldanamycin (GA). The anti-cancer efficacy of conjugates was evaluated in HCC cell cultures with and without hyperthermia (43?°C). The conjugates were also administered twice weekly in a murine HCC model as a single treatment or in combination with single electrocautery as the ablation method. Results: ELP-GA conjugates displayed enhanced cytotoxicity in vitro and effective heat shock inhibition under hyperthermia. The conjugates alone significantly slowed the tumour growth without systemic toxicity. Four doses of thermo-responsive ELP-GA conjugates with concomitant simple electrocautery accomplished significant Hsp90 inhibition and sustained tumour suppression. Conclusion: Hsp90 inhibition plays a key role in preventing the recurrence of HCC, and the combination of ablation with targeted therapy holds great potential to improve prognosis and survival of HCC patients.     

Effect of thermal dose on heat shock protein expression after radio-frequency ablation with and without adjuvant nanoparticle chemotherapies

Purpose: The aim of this study was to evaluate the effect of different radio-frequency ablation (RFA) thermal doses on coagulation and heat shock protein (HSP) response with and without adjuvant nanotherapies.

Materials and methods: First, Fischer rats were assigned to nine different thermal doses of hepatic RFA (50–90?°C, 2–20?min, three per group) or no treatment (n?=?3). Next, five of these RF thermal doses were combined with liposomal-doxorubicin (Lipo-Dox, 1?mg intravenously) in R3230 breast tumours, or no tumour treatment (five per group). Finally, RFA/Lipo-Dox was given without and with an Hsp70 inhibitor, micellar quercetin (Mic-Qu, 0.3?mg intravenously) for two different RFA doses with similar coagulation but differing peri-ablational Hsp70 (RFA/Lipo-Dox at 70?°C × 5?min and 90?°C × 2?min, single tumours, five per group). All animals were sacrificed 24?h post-RFA and gross tissue coagulation and Hsp70 (maximum rim thickness and % cell positivity) were correlated to thermal dose including cumulative equivalent minutes at 43?°C (CEM₄₃).

Results: Incremental increases in thermal dose (CEM₄₃) correlated to increasing liver tissue coagulation (R² = 0.7), but not with peri-ablational Hsp70 expression (R² = 0.14). Similarly, increasing thermal dose correlated to increasing R3230 tumour coagulation for RF alone and RFA/Lipo-Dox (R² = 0.7 for both). The addition of Lipo-Dox better correlated to increasing Hsp70 expression compared to RFA alone (RFA: R² = 0.4, RFA/Lipo-Dox: R² = 0.7). Finally, addition of Mic-Qu to two thermal doses combined with Lipo-Dox resulted in greater tumour coagulation (p?<?0.0003) for RFA at 90?°C × 2?min (i.e. greater baseline Hsp70 expression) than an RFA dose that produced similar coagulation but less HSP expression (p?<?0.0004).

Conclusion: Adjuvant intravenous Lipo-Dox increases peri-ablational Hsp70 expression in a thermally dependent manner. Such expression can be exploited to produce greater tumour destruction when adding a second adjuvant nanodrug (Mic-Qu) to suppress peri-ablational HSP expression.     

Heat shock proteins in hepatocellular carcinoma: Molecular mechanism and therapeutic potential

Heat shock proteins (HSPs) are highly conserved proteins, which are expressed at low levels under normal conditions, but significantly induced in response to cellular stresses. As molecular chaperones, HSPs play crucial roles in protein homeostasis, apoptosis, invasion and cellular signaling transduction. The induction of HSPs is an important part of heat shock response, which could help cancer cells to adapt to stress conditions. Because of the constant stress condition in tumor microenvironment, HSPs overexpression is widely reported in many human cancers. In light of the significance of HSPs for cancer cells to survive and obtain invasive phenotype under stress condition, HSPs are often associated with poor prognosis and treatment resistance in many types of human cancers. It has been described that upregulation of HSPs may serve as diagnostic and prognostic markers in hepatocellular carcinoma (HCC). Targeting HSPs with specific inhibitor alone or in combination with chemotherapy regimens holds promise for the improvement of outcomes for HCC patients. In this review, we summarize the expression profiles, functions and molecular mechanisms of HSPs (HSP27, HSP70 and HSP90) as well as a HSP‐like protein (clusterin) in HCC. In addition, we address progression and challenges in targeting these HSPs as novel therapeutic strategies in HCC.     

Expression of 70-kDa heat shock protein in oral lesions: marker of biological stress or pathogenicity

We showed differential expression of HSP70 during oral tumorigenesis. The precise functional role of HSP70 overexpression in the pathogenesis of betel and tobacco related oral cancer remains to be determined. To evaluate the utility of HSP70 as an indicator of the biological stress experienced by tumour cells or the malignant potential of oral epithelial lesions and predicting clinical outcome, its expression was assessed in different stages of oral carcinogenesis by immunohistochemical analysis and correlated with clinicopathological parameters. Overexpression of HSP70 protein was observed in 38 of 64 (59%) dysplastic lesions and 92 of 125 (74%) oral squamous cell carcinomas (SCCs) which included 76 of 105 cases (72%) of primary oral SCCs and 16 of 20 (80%) of recurrent oral SCCs. A significant correlation of HSP70 expression was observed with severity of dysplasia (P=0.0006767), poor histological differentiation of primary tumours (P=0.0184348), increase primary tumour size (P=0.0221103) and consumption of betel and tobacco (P<0.01). Follow-up studies showed that in patients with premalignant lesions the median transition time (premalignancy to malignancy) was significantly shorter in HSP70 overexpressing cases than those showing basal level of HSP70 (P=0.012). Oral cancer patients with elevated levels of HSP70 showed decreased median disease-free survival time (no recurrence/metastasis) than those showing basal HSP70 immunoreactivity (P=0.0246). The results suggest that HSP70 expression may not be a mere marker of biological stress but may also be implicated in the pathogenesis of oral cancer.     

Heat shock protein inhibitor,quercetin, as a novel adjuvant agent to improve radiofrequency ablation-induced tumor destruction and its molecular mechanism

Background

We investigated the effect of a small molecular inhibitor of heat shock protein (HSP), quercetin, on tumor radiofrequency (RF) ablation, and explored the underlying molecular mechanisms.

Methods

In in vivo study, rats with R3230 breast adenocarcinoma were sacrificed 24 h post-treatment and gross coagulation areas were compared, and next, randomized into four treatment arms (control, quercetin alone, RF alone, and combination) for Kaplan-Meier analysis of defined endpoint survival. Then the distribution and expression levels of heat shock protein 70 (HSP70), cleaved caspase-3 and heat shock factor 1 (HSF1) were analyzed after different treatments. In in vitro study, we used quercetin to promote SK-HEP-1 (hepatic) and MCF-7 (breast) cancer cell apoptosis in heat shock cell model, and siRNA was used to block c-Jun and to explore the role of activating protein-1 (AP-1) signaling pathways.

Results

We found the effects of quercetin plus RFA resulted in increase on the tumor destruction/endpoint survival (26.5±3.4 d) in vivo, compared with RF alone (17.6±2.5 d) and quercetin alone (15.7±3.1 d). Most importantly, quercetin-induced cancer cell death required the presence of HSF1 in animal model. Furthermore, quercetin directly down-regulated expression of HSF1 in vitro, which our findings have revealed, required the activation of AP-1 signaling pathways by loss-of-function analysis using siRNA mediated targeting of c-Jun.

Conclusions

These results indicated a protective role of quercetin in tumor ablation and highlighted a novel mechanism involving HSP70 with HSF1 pathway in thermal ablation of solid tumors.     

Radiofrequency ablation combined with liposomal quercetin to increase tumour destruction by modulation of heat shock protein production in a small animal model

Purpose: To investigate the effect of heat shock protein (HSP) modulation on tumour coagulation by combining radiofrequency (RF) ablation with adjuvant liposomal quercetin and/or doxorubicin in a rat tumour model.

Methods: Sixty R3230 breast adenocarcinoma tumours/animals were used in this IACUC-approved study. Initially, 60 tumours (n?=?6, each subgroup) were randomised into five groups: (1) RF alone, (2) intravenous (IV) liposomal quercetin alone (1?mg/kg), (3) IV liposomal quercetin followed 24?h later with RF, (4) RF followed 15?min later by IV liposomal doxorubicin (8?mg/kg), (5) IV liposomal quercetin 24?h before RF followed by IV liposomal doxorubicin 15?min post-ablation. Animals were sacrificed 4 or 24?h post-treatment and gross coagulation diameters were compared. Next, immunohistochemistry staining was performed for Hsp70 and cleaved caspase-3 expression. Comparisons were performed by using Student t-tests or ANOVA.

Results: Combination RF-quercetin significantly increased coagulation size compared with either RF or liposomal quercetin alone (13.1?±?0.7?mm vs. 8.8?±?1.2?mm or 2.3?±?1.3?mm, respectively, P?<?0.001 for all comparisons). Triple therapy (quercetin-RF-doxorubicin) showed larger coagulation diameter (14.5?±?1.0?mm) at 24?h than quercetin-RF (P?=?0.016) or RF-doxorubicin (13.2?±?1.3?mm, P?=?0.042). Combination quercetin-RF decreased Hsp70 expression compared with RF alone at both 4?h (percentage of stained cells/hpf 22.4?±?13.9% vs. 38.8?±?16.1%, P?<?0.03) and 24?h (45.2?±?10.5% vs. 81.1?±?3.6%, P?<?0.001). Quercetin-RF increased cleaved caspase-3 expression at both 4?h (percentage of stained cells/hpf 50.7?±?13.4% vs. 41.9?±?15.1%, P?<?0.03) and 24?h (37.4?±?7.8% vs. 33.2?±?6.5%, P?=?0.045); with, triple therapy (quercetin-RF-doxorubicin) resulting in the highest levels of apoptosis (45.1?±?10.7%) at 24?h. Similar trends were observed for rim thickness.

Conclusions: Suppression of HSP production using adjuvant liposomal quercetin can increase apoptosis and improve RF ablation-induced tumour destruction. Further increases in tumour coagulation can be seen including an additional anti-tumour adjuvant agent such as liposomal doxorubicin.     

热休克蛋白与肿瘤免疫

热休克蛋白(heat shock protein,HSP)是一类在生物进化中高度保守、广泛存在于原核及真核生物中的蛋白质。近年热休克蛋白在免疫中的作用已成为当前研究的热点之一。已证实其能与肿瘤细胞内多肽分子结合,通过抗原提呈细胞上的受体,将抗原肽传递给细胞毒Ｔ细胞诱导特异性抗肿瘤免疫应答。热休克蛋白肽复合物作为一种疫苗,在生物治疗方面拥有广阔的治疗前景,值得深入研究。     

Role of Pluronic block copolymers in modulation of heat shock protein 70 expression

Purpose: The goal of this study was to evaluate the relationship between previously demonstrated thermosensitising effects of the block copolymer, Pluronic, and heat shock protein 70 (Hsp70) expression in an experimental colorectal cancer model in vitro and in vivo.

Materials and methods: Rat colorectal carcinoma cells were treated with low-grade hyperthermia (43°C) alone or in combination with Pluronics L10 (3?mg/mL), L61 (0.3?mg/mL), or L64 (0.5?mg/mL) for 20?min. Adinosine triphosphate (ATP) levels and cell viability were determined using standard assays. Hsp70 expression was quantified by western blot for cells treated with L10, L61, and L64 at doses specified above and Pluronic P85 (10?mg/mL) alone and in combination with heat. BDIX rats with flank tumours were used to study the effect of L61 and hyperthermia on Hsp70 expression in vivo.

Results: In vitro, treatment with L10, L61, and L64 plus low-grade hyperthermia lead to depletion of ATP levels to between 8 and 66% of untreated control after 24 h. Maximum expression of Hsp70 was observed at 9?h following hyperthermia alone. The combination of low-grade hyperthermia and Pluronic treatment reduced Hsp70 expression for up to 6 hours, and L10 appeared to completely inhibit the Hsp70 expression. In vivo, Hsp70 expression was increased 5?h after hyperthermia in BDIX rat tumour models and no Hsp70 expression was observed in L61 pre-treated and control groups.

Conclusion: Pluronic effectively improves hyperthermic and low-grade hyperthermic treatment in part due to reduction of Hsp70 expression.     

胸腺肽α1对肝细胞癌患者热消融术后免疫功能及预后的影响

目的:探讨胸腺肽α1对原发性肝癌患者热消融术后免疫功能及预后的影响。方法:回顾性收集2010年至2012年在河南中医药大学第一附属医院的BCLC-A期原发性肝癌患者热消融术后共80例,按热消融术后是否使用胸腺肽α1分为观察组及对照组（各40例）,比较两组患者的免疫功能及远期治疗效果。结果:与对照组相比,观察组消融1年后仍存在较高的CD4+/CD8+比值（1.34±0.36 vs 0.64±0.26,P＜0.01)。观察组1年生存率、3年生存率及5年生存率分别为97.5%、90.0%及75.0%,对照组1年生存率、3年生存率及5年生存率分别为97.5%、80.0%及57.5%,二者的5年生存率比较差异有统计学意义（P=0.09）。观察组1年无瘤生存率、3年无瘤生存率及5年无瘤生存率分别为97.5%、72.5%及47.5%,对照组1年无瘤生存率、3年无瘤生存率及5年无瘤生存率分别为95.0%、52.5%及27.5%,二者的5年无瘤生存率比较差异有统计学意义（P=0.06）。结论:胸腺肽α1能改善原发性肝癌热消融后患者的CD4+/CD8+比值,提高患者的无瘤生存率及总生存率。     

热休克蛋白27基因在食管鳞状上皮癌变过程中的表达

目的:研究食管鳞癌、癌旁及正常食管粘膜中热休克蛋白27(HSP27)基因的表达。方法:采用RTPCR方法,定量检测26例食管鳞癌、癌旁及正常食管粘膜中HSP27基因表达水平。结果:以HSP27/GAPDHmRNA灰度比值表示HSP27基因的相对表达量。食管鳞癌、癌旁(不典型增生)和正常食管粘膜中的HSP27/GAPDH灰度比值分别为0.81±0.27、0.90±0.24和1.22±0.39。HSP27基因表达水平在食管鳞癌和不典型增生食管粘膜中的表达量明显低于正常食管粘膜(P<0.05),而HSP27基因在食管鳞癌和不典型增生食管粘膜中的表达量差别无显著性(P>0.05)。食管鳞癌、不典型增生食管粘膜及正常食管粘膜中的HSP27基因表达在不同患者年龄、性别和淋巴结转移之间均无显著性差异(P>0.05)。结论:HSP27基因在食管鳞癌和不典型增生食管粘膜中表达与正常食管粘膜相比明显降低,提示HSP27基因在食管鳞癌的发生、发展过程中有不同程度的表达缺失。因此,提高HSP27基因在食管鳞癌中的表达将来可能会成为一种非常有效的生物学治疗手段。     

Percutaneous thermal ablation of medium and large hepatocellular carcinoma

BACKGROUND:

Radiofrequency ablation (RFA) and microwave ablation (MWA) were found to be effective in treating hepatocellular carcinoma (HCC) smaller than 3 cm; however, to the authors' knowledge, the usefulness of thermal ablation in treating larger HCC, especially those >5 cm, has not been well documented. The present study evaluated the therapeutic efficacy of percutaneous thermal ablation with curative intention for HCC measuring between 3.0 cm and 7.0 cm.

METHODS:

Percutaneous RFA or MWA were used to treat 109 HCC patients with at least 1 tumor measuring between 3.0 cm and 7.0 cm. Fifty?eight patients received thermal ablation as the first treatment, and the remaining 51 were treated for posthepatectomy recurrent HCC. A total of 89 patients had a main tumor measuring 3.0 cm to 5.0 cm, and 20 patients had main tumors measuring 5.0 cm to 7.0 cm. Local therapeutic efficacy, long‐term outcome, and prognostic factors were analyzed.

RESULTS:

There were no treatment‐related deaths, and the major complication rate was 9.2%. Complete ablation rate was 92.6%. Local recurrence (LR) occurred in 22% patients, with a median time to LR of 4.6 months. Distant recurrences developed in 53.2% patients. The 1‐year, 3‐year, and 5‐year survival rates were 75.8%, 30.9%, and 15.4%, respectively. Univariate analysis indicated that incomplete tumor ablation, posthepatectomy recurrence, and preablation α‐fetoprotein (AFP) ≥200 ng/mL were 3 unfavorable prognostic factors for long‐term survival (P = .000, .015, and .008, respectively). Cox regression analysis confirmed that incomplete tumor ablation, recurrent tumors, and preablation AFP ≥200 ng/mL were independent unfavorable prognostic factors, with an exp(B) of 4.158 (P = .001), 1.568 (P = .082), and 1.593 (P = .082), respectively.

CONCLUSIONS:

Percutaneous thermal ablation was effective and safe in treating HCC between 3 cm and 7 cm, with acceptable local tumor control and long‐term outcomes. Completeness of ablation, previous history of treatment, and preablation AFP level were significant prognostic factors. Cancer 2009. © 2009 American Cancer Society.     

结肠癌中热休克蛋白70和糖调节蛋白94的表达与临床病理的关系

目的:探讨热休克蛋白70(HSP70)和糖调节蛋白94(grp94)在人结肠癌组织中的表达与临床病理的关系。方法:应用免疫组织化学和病理学图像分析方法研究80例人结肠癌组织及其癌旁组织、发生及未发生转移的癌组织中HSP70和grp94的表达。结果:在结肠癌组织中HSP70和grp94的表达明显高于癌旁组织(92.5%,85.0%VS56.3%,42.5%,P<0.01)。HSP70和grp94在中度和低度分化的结肠癌组织中的表达率明显高于癌旁组织(93.7%,87.5%;100%,90.0%VS56.3%,42.5%;P<0.01)。HSP70和grp94在DukesC期(97.1%,91.2%)和D分期(100%,90.9%)的表达比DukesA期(80.0%,70.0%)和B期(78.6%,71.4%)的癌组织阳性表达率高(P<0.05)。结论:HSP70和grp94在发生转移的低分化结肠癌组织和未发生转移的高分化癌组织中表达存在明显不同,它们的高表达可以作为结肠癌的诊断或预后指标。     

热休克蛋白HDJ1影响肺腺癌细胞侵袭和迁移能力的研究

目的：研究热休克蛋白HDJ1在人肺腺癌组织、肺腺癌细胞株以及正常肺上皮细胞株中的表达情况,探讨HDJ1对肺腺癌细胞侵袭和迁移能力的影响。方法：采用免疫组织化学法检测HDJ1在人肺腺癌组织和癌旁正常组织中的表达情况;Western blot检测HDJ1在人肺腺癌细胞A549、H1299、H292及正常肺上皮细胞Bease-2B中的表达情况;使用pMagic 7.1-HDJ1-KD慢病毒转染A549细胞构建HDJ1稳定下调表达的细胞模型,并通过划痕实验、Transwell实验检测细胞的侵袭和迁移能力。结果：HDJ1在人肺腺癌组织中呈高表达;HDJ1在A549细胞中呈高表达;细胞划痕实验结果显示,与对照组细胞比较,HDJ1表达下调的A549细胞迁移率下降（P < 0.05）;Transwell实验结果表明,相对于对照组细胞,HDJI表达下调的A549细胞在侵袭和迁移实验中的穿膜细胞数均减少（P < 0.05）。结论：HDJ1在肺腺癌组织和细胞中均呈高表达,并促进肺腺癌细胞的侵袭和迁移能力。     

人胃癌组织中热休克蛋白70和糖调节蛋白94的表达及其意义

目的：探讨休克蛋白70（HSP70）和糖调节蛋白94（grp94）在人胃癌组织中的表达与临床病理的关系。方法：应用免疫组织化学和病理学图像分析方法研究60例人胃癌组织及其癌旁组织、发生及未发生转移的癌组织中HSP70和grp94的表达。结果：在胃癌组织中HSP70和grp94的表达明显高于癌旁组织（93.3%,81.7%vs36.7%,25.0%,P〈0.01）。HSP70和grp94在低分化和发生转移的胃癌组织中的表达明显高于非转移癌和癌旁组织（90.0%,85.0%;100%,84.6%vs36.7%,25.0%;P〈0.01）。结论：HSP70和grp94在发生转移的低分化胃癌组织和未发生转移的高分化癌组织中表达存在明显不同,可以作为胃癌的诊断或预后指标。     

Microwave ablation versus radiofrequency ablation for the treatment of hepatocellular carcinoma: A systematic review and meta-analysis

Purpose: Radiofrequency ablation (RFA) and microwave ablation (MWA) are the two main percutaneous techniques for the treatment of unresectable hepatocellular carcinoma (HCC). However, to date, studies comparing the two therapies have provided discordant results. The aim of this meta-analysis is to evaluate the efficacy and safety of the two treatments for HCC patients. Materials and methods: A computerised bibliographic search was performed on PubMed/MEDLINE, Embase, Google Scholar and Cochrane library databases. The rates of complete response (CR), local recurrence (LRR), 3-year survival (SR) and major complications were compared between the two treatment groups by using the Mantel-Haenszel test in cases of low heterogeneity or the DerSimonian and Laird test in cases of high heterogeneity. Sources of heterogeneity were investigated using subgroup analyses. In order to confirm our finding, sensitivity analysis was performed restricting the analysis to high-quality studies. Results: One randomised controlled trial (RCT) and six retrospective studies with 774 patients were included in the meta-analysis. A non-significant trend of higher CR rates in the patients treated with MWA was found (odds ratio (OR)?=?1.12, 95% confidence interval (CI) 0.67–1.88, p?=?0.67]. Overall LRR was similar between the two treatment groups (OR 1.01, 95% CI 0.53–1.87, p?=?0.98) but MWA outperformed RFA in cases of larger nodules (OR 0.46, 95% CI 0.24–0.89, p?=?0.02). 3-year SR was higher after RFA without statistically significant difference (OR 0.95, 95% CI 0.58–1.57, p?=?0.85). Major complications were more frequent, although not significantly, in MWA patients (OR 1.63, 95% CI 0.88–3.03, p?=?0.12). Conclusions: Our results indicate a similar efficacy between the two percutaneous techniques with an apparent superiority of MWA in larger neoplasms.     

热休克蛋白 90--癌症治疗的新靶点

热休克蛋白90(heat shock protein90,Hsp90)作为分子伴侣参与调控、维持细胞内多种蛋白的构象和功能,以帮助细胞在应激环境刺激下正常生长。近年来研究表明很多癌基因蛋白均为Hsp90的作用靶点,因此抑制Hsp90的功能将促进这些癌基因蛋白的降解,有助于癌症治疗。体内外实验也证实了Hsp90抑制剂的抗肿瘤活性,其中17-烯丙胺-17-脱甲氧格尔德霉素(17-allylamio-17-desmethoxygeldanamycin,17-AAG)正在进行临床试验。