Therapeutic Effects of Radiolabeled 17-Allylamino-17-Demethoxygeldanamycin on Human H460 Nonsmall-Cell Lung Carcinoma Xenografts in Mice

Abstract

Heat shock protein 90 (HSP90) is an exciting molecular target for cancer therapy because of this protein's key role in oncogenic signaling pathways. In this work, the binding property of 17-allylamino-17-demethoxygeldanamycin (17-AAG), an HSP90 inhibitor now in phase II clinical trials, was used by labeling with radioisotope iodine-131 (¹³¹I), to observe the potential therapeutic effects on nonsmall-cell lung carcinoma (NSCLC) xenografts. The compound ¹³¹I-17-AAG and BALB/c nude mice bearing H460 human NSCLC xenografts were prepared. Intratumoral and intravenous administration routes were used. The potential effects of labeled 17-AAG were evaluated by biodistribution studies, in vivo imaging, cancer-treatment studies, and histological analysis. Specific tumor uptake of ¹³¹I-17-AAG was achieved in the xenograft models. Compared to intravenous (i.v.) application, tumor uptake was significantly improved with intratumoral injection of ¹³¹I-17-AAG and competitively reduced with preinjection of unlabeled 17-AAG. All treatment groups established tumor-growth inhibition compared with the control group (p < 0.05), with a certain dose-dependent relationship. The 16-day inhibition ratios via intratumoral delivery were better than those via i.v. delivery (p < 0.05). No significant treatment-induced abnormalities appeared in the mice. Compared with the control group, HSP90α + expression and Ki-67+ expression of tumor tissues declined after treatments (p < 0.05), which correlated positively with tumor inhibition. Thus, ¹³¹I-17-AAG treatment undoubtedly had inhibitive effects on human NSCLC xenografts in mice, and the combination of radionuclides and HSP90 inhibitors may be considered for cancer therapy.

Introduction

During the last few decades, anticancer drugs had to face the genetic plasticity of cells producing new drug-resistant cancers. Heat shock protein 90 (HSP90) is a molecular chaperone that belongs to an ancient, well-conserved class of proteins. It plays a key role in the conformational maturation of oncogenic signaling proteins^1
–3 such as HER-2/Neu (ErbB2), Akt, Raf-1, Bcr-Abl, mutated p53, and HIF-1α.^4,5 Inhibition of HSP90 leads to depletion of these oncogenic clients, via the ubiquition proteasome pathway, and delivers a simultaneous combinatorial attack on all the hallmark traits of malignancy.⁶ Therefore, inhibition of HSP90 represents an exciting target for antitumor therapy.⁷

17-Allylamino-17-demethoxygeldanamycin (17-AAG) is the first-in-class HSP90 inhibitor in clinical trials. A derivative of geldanamycin, 17-AAG has important biological activities but less toxicity.⁸ Phase I clinical trials^9
–11 with 17-AAG have shown that the drug itself is not associated with the expected therapeutic responses, but is systemically well-tolerated and provides a protracted stabilization of disease. Yet, 17-AAG chemotherapy still lacks direct means of observation. The best strategy to make use of the action of HSP90 inhibitors for anticancer therapy remains to be defined.

HSP90 accounts for 1%–2% of all cellular proteins in most mammalian cells,¹² but its inhibitors kill cancer cells selectively compared with normal cells.^13,14 Tumor HSP90 is present entirely in multichaperone complexes with high ATPase activity, whereas HSP90 from normal tissues is in a latent, uncomplexed state.¹⁵ Kamal et al. reported that HSP90 derived from tumor cells has a 100-fold higher binding affinity for 17-AAG than does HSP90 from normal cells.¹⁶ Therefore, modulation of HSP90 function by 17-AAG targeting has emerged as a candidate for molecular cancer therapy.¹⁷

Carrier-based radionuclide therapy is highly effective, requires short treatment time, and is considerably less toxic compared with chemotherapy.¹⁸ Radiolabeled agents are also far more efficient than their corresponding unlabeled agents. This work made use of the binding property of 17-AAG, labeled with a radionuclide, to observe the potential therapeutic effects of this combination. Radioisotope iodine-131 (¹³¹I) was selected here as the radiolabeled substance for several reasons: (1) it is one of the most commonly used therapeutic radioisotopes in China; (2) it is inexpensive; (3) it has a short ß-particle path length of ¹³¹I (typically about 1 mm), so the tumor foci could be treated more safely and effectively; (4) γ-emission of ¹³¹I allows noninvasive imaging, though with some limitations as a result of its 364 keV energy; (5) the 8-day half-life of ¹³¹I is well-matched to ¹³¹I's relatively slow biologic localization; and (6) free iodine is substantially excreted through the kidneys in patients on thyroid blockade in advance,¹⁹ as thyroid uptakes free iodine significantly, affecting the results of imaging. Nonsmall-cell lung carcinoma (NSCLC) is the most common cancer and is the major killer throughout the world.²⁰ HSP90 inhibitors could be evaluated in multiple lung-cancer subsets.^21,22 The H460 human NSCLC tumor model was chosen as the object of this study.

Materials and Methods

General

All reagents were obtained from commercial sources.

Radiolabeling

17-AAG (NSC 330507) purchased from Sigma-Aldrich Corporation was dissolved in ethanol absolute to yield 10 mg/mL and stored at −20°C. Na¹³¹I solution used in these procedures was obtained from Sengke Pharmaceuticals Ltd. The synthesis of ¹³¹I-AAG has been described as previously reported.²³ 17-AAG (17 nmol, 1.0 mg/mL of 95% ethanol) was mixed with 4 M HCL, Na¹³¹I (185 MBq), and 5% hydrogen peroxide, and incubated at 40°C for 10 minutes. The reaction was terminated by excessive 3 M sodium metabisulfite and adjusted to an appropriate pH (7.0–7.4) by adding 4 M ammonium hydroxide. The product was purified by high-performance liquid chromatography (HPLC) using a Hyrersil C18 column (4.6 × 200 mm, 5 μm; Elite) with 50% (v/v) 25 mM sodium phosphate (pH 3.00), 10 mM triethylamine, and 50% acetonitrile, and sterilized by using a 0.2 μm filter. The purified solution was dried under nitrogen atmosphere and diluted in saline and anhydrous ethanol (5:1, v/v) before use. In vitro stability of the labeled compound was evaluated in the saline or rat serum at 37°C for 120 hours. The radiochemical purity of ¹³¹I-AAG analyzed by thin layer chromatography (TLC), developed with dichloromethane and methanol (9:1, v/v), was above 98% immediately after synthesis and above 91.8% 120 hours after purification. The R_f value was 0.6–0.9. The specific activity of ¹³¹I-AAG was 148 GBq/μmol for all experiments.

Cell lines and tumor xenografts

The H460 human NSCLC cell lines were obtained from the Cell Resource Center of the Chinese Academy of Sciences and were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. Four (4)- to 6-week-old female BALB/c athymic nude mice (18–20 g) were purchased from the Shanghai Cancer Research Institute and fed with sterilized pelleted food and sterilized water. These mice were maintained free of specific pathogens throughout the study period. Experiments with animals were conducted in accordance with relevant institutional guidelines, and the experimental procedures were approved by the Southeast University Animal Care and Use Committee.

Tumor cells to be implanted into the mice were harvested to near confluence by incubation with 0.25% trypsin-ethylenediaminetetraacetic acid. The cells were pelleted by centrifugation at 1000 rpm for 5 minutes and resuspended in sterile phosphate-buffered saline (PBS). Cells (1 × 10⁷/mouse) were implanted subcutaneously (s.c) into the right flank of female nude mice. The mice were used when the tumor maximum diameter reached 8–10 mm (about 2 weeks after implantation).

All the model mice were fed with sterilized 1% potassium perchlorate water solution for 3 days before all injections, to determine thyroid blockade in advance. Before imaging, the mice were anesthetized with a single dose of 50 mg/kg body weight of pentobarbital injected intraperitoneally (i.p).

Biodistribution studies

For biodistribution studies, 24 nude mice bearing the H460 tumor were randomly assigned into groups of four. ¹³¹I-17-AAG was injected intratumorally or intravenously at a dose of 1.85 MBq in 100 μL sterile solution. The mice in each group were sacrificed and dissected at 0.5, 4, and 24 hours postinjection. Tumors and major organs were collected and wet-weighted. To measure plasma radioactivity, 50 μL of whole blood was withdrawn from the tail veins at 0.5, 1, 4, 8, and 24 hours using capillary tubes. The samples were counted in a γ counter. The data were expressed as percentage injected dose per g of tissue (% ID/g) and as the percentage injected dose per mL of blood (% ID/mL). For each mouse, the radioactivity in the tissue samples was calibrated against a known quantity of the injected dose and normalized based upon the body weight of the mouse. Values were expressed as mean ± standard deviation (SD) for each group.

In vivo imaging

Sixteen (16) tumor-bearing nude mice were randomly divided into groups of four, including the ¹³¹I-AAG intratumoral injection group, the ¹³¹I-AAG tail vein injection group, the Na¹³¹I intratumoral injection group, and the blocking group. Na¹³¹I solution was added with ethanol (1:5, v/v) to make its formulation similar to that of ¹³¹I-17-AAG solution, and so was unlabeled 17-AAG solution. The intratumoral injection was performed with a 1 mL syringe containing the radioactive tracer. The needle was carefully introduced into the tumor and withdrawn slowly while releasing the tracer into the tumor. The aim was to maintain an even distribution of the tracer within the tumor and prevent any outflow. Each mouse in the first three groups received a radioactivity dose of 5.55 MBq per 100 μL per injection. Five (5)-minute planar static images in prone position were acquired at 30 minutes, 4 hours, 24 hours, 2 days, 5 days, and 7 days after injection, using a γ camera (Millennium VG Hawkeye GE Medical System) equipped with a pinhole collimator (magnification factor of 3.8 for a crystal-to-object distance of 3.0 cm). A 20% window around the 364 keV photopeak was used here. Graphic processing software (Hawkeye applications software) was used for data analysis. For the blocking study, tumor-bearing mice were each given intravenous (i.v.) injections of 200 μg (about 340 nmol) of 17-AAG 2 hours before the intratumoral injections of 5.55 MBq (about 37.5 pmol) of ¹³¹I-17-AAG. Images were acquired 0.5, 4, and 24 hours after tracer injection with the aforementioned imaging setting. All the mice were sacrificed after imaging. Tumors and major organs were removed for radioactivity measurement and histopathologic analysis. Tumor radioactivity was expressed as % ID/g.

Cancer-treatment studies

Antitumor efficacy of radiolabeled 17-AAG was investigated in mice models bearing H460 tumor xenografts. The model mice were prepared as described above and randomized into seven groups of 4 mice each. Dosage was set up based on the human safe-dose range for ¹³¹I delivery. Groups 1–3 received intratumoral injections of 0.1 mL of ¹³¹I-17-AAG at doses of 5.5 MBq × 2 at an 8-day interval, 11.0 MBq × 1, and 5.5 MBq × 1, respectively. Groups 4–6 received the same doses of ¹³¹I-17-AAG via tail-vein injection. The four tumor-bearing mice in Group 7 were used as control. Regular observations were made before and after administration. Tumor volumes were assessed every 2 days after injection by external vernier caliper measurements along the longest axis (x-axis) and the axis perpendicular to it (y-axis), and then calculated by using the following formula: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\rm Volume} = 4/3 \cdot \pi \times (x/2)^2 \times (y/2). \end{align*} \end{document}

All mice were sacrificed at 16 days after injection. The tumors were dissected and weighted. Tumor inhibition ratios were calculated using the following formula: \documentclass{aastex} \usepackage{amsbsy} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{bm} \usepackage{mathrsfs} \usepackage{pifont} \usepackage{stmaryrd} \usepackage{textcomp} \usepackage{portland, xspace} \usepackage{amsmath, amsxtra} \pagestyle{empty} \DeclareMathSizes{10}{9}{7}{6} \begin{document} \begin{align*}{\hbox {\rm Inhibition ratio}} = [({\rm W}1 - {\rm W}2)/{\rm W}1] \times 100 \%; \end{align*} \end{document}

with W1 being the average tumor mass of the control group and W2 being the tumor mass of each mouse in each treatment group.

The tumor tissues and major organs were harvested and placed in 10% formalin for a minimum of 48 hours. Trimmed organs were sent to a pathology laboratory, where they were embedded in paraffin, sectioned (5 μm thickness), and stained with hematoxylin and eosin (H&E).

Immunohistochemical staining

All paraffin sections were sent to the pathology laboratory of a research center and stained following a routine EnVision two-step staining method. Before immunohistochemical (IHC) staining, these tumor tissue slices (5 μm thick) were microwave-treated to retrieve antigen epitopes. After rinsing with PBS and blocking with 10% donkey serum for 30 minutes at room temperature, the slices were incubated with goat antihuman HSP90α antibody (1:500; Santa Cruz Biotechnology) and rabbit antihuman Ki-67 antibody (1:200; Fuzhou Maxim Biotechnology) for 1 hour at room temperature. After incubation, the primary antibody was washed away three times with PBS. The secondary antibody biotinylated IgG was pipetted onto the sections and incubated for 20 minutes at room temperature. After rinsing for 5 minutes in PBS three times, the final staining was done in diaminobenzidine tetrahydrochloride solution and H&E solution. Extra stain was washed away with PBS. The slides were then transferred through an ascending ethanol series, finally through xylene, and then mounted.

The area with the highest fraction of stained cells in the sections was chosen using a 10× objective magnification. Then, a 20× objective was used to count arbitrarily the fraction of positive stained cells among 200 cancer cells. In this study, the cells were counted from the infiltrating part of the cancer tissue. A cell was considered as Ki-67+ if there was a clearly detectable brown color inside the nucleus, and as HSP90α+ if there was a clearly detectable brown color mainly in the cytoplasm. The fraction of positively stained cells was called the HSP90α+ fraction or the Ki-67+ fraction in this study.

Statistical analysis

SPSS 13.0 software was used for statistical analysis. Quantitative data were expressed as mean ± SD. Means were compared using one-way analysis of variance (ANOVA) and the Student t-test. If one-way analysis of variance (ANOVA) was significant, pair-wise comparisons were made using the Student-Newman-Keuls test. The Pearson correlation coefficient was used to evaluate correlation among the HSP90α+ and Ki-67+ fractions. p-Values less than 0.05 were considered statistically significant.

Results

Biodistribution studies

The biodistribution studies on the effects of ¹³¹I-17-AAG administered via intratumoral or intravenous routes were performed in nude mice bearing tumors derived from the H460 human NSCLC cell lines. The radioactivity changes in the blood are shown in Figure 1. The intratumoral group had a lower accumulation of radioactivity in the blood pool, with a 4-hour peak value of 5.17 ± 0.70% ID/mL and a low 24-hour value of 0.35 ± 0.04% ID/mL, relative to 26.78 ± 6.03% ID/mL at 0.5 hour and 4.23 ± 1.33% ID/mL at 24 hours postinjection, respectively, for the i.v. group (p < 0.05). The tumor uptake via i.v. delivery was relatively high (13.87 ± 8.07% ID/g at 24 hours postinjection) compared with the uptake in normal organs, but was significantly lower than that via intratumoral delivery (121.10 ± 31.63% ID/g at 24 hours postinjection; p < 0.05) (Fig. 2). The biodistribution in major organs and tissues over 24 hours is shown in Figure 3. The radioactivity in normal organs was less than or around 1% ID/g at 24 hours after injection, which revealed a rapid systemic clearance. The low accumulation of radioactivity in the stomach also indicated a low dehalogenation of the tracer.

FIG. 1.

Graphical comparison of plasma radioactivity at different time points over 24 hours after (A) intratumoral or (B) intravenous injection. The H460 tumor-bearing mice were treated with 1.85 MBq of ¹³¹I-17-AAG per mouse. Columns, mean plasma radioacitivity (% ID/mL); bars, SD (n = 4). 17-AAG, 17-allylamino-17-demethoxygeldanamycin; % ID/mL, percentage injected dose per mL of blood; SD, standard deviation.

FIG. 2.

Graphic representation of changes in tumor uptake at 0.5, 4, and 24 hours after (A) intratumoral and (B) intravenous injection. The H460 tumor-bearing mice were treated with 1.85 MBq ¹³¹I-17-AAG per mouse. Columns, mean % ID/g tumors; bars, SD (n = 4). 17-AAG, 17-allylamino-17-demethoxygeldanamycin; % ID/g, percentage injected dose per g of tissue; SD, standard deviation.

FIG. 3.

Biodistribution in major organs and tissues over 24 hours, after administration of 1.85 MBq ¹³¹I-17-AAG per mouse. (A) Intratumoral delivery, (B) intravenous delivery. Columns, mean % ID/g of major organs or tissues; bars, SD (n = 4). 17-AAG, 17-allylamino-17-demethoxygeldanamycin; % ID/g, percentage injected dose per g of tissue; SD, standard deviation.

In vivo imaging analysis

The tumor localization and organ distribution of intratumorally and i.v. applied ¹³¹I-17-AAG were compared, using an unlabeled ¹³¹I radionuclide or a blocking study as a control. Representative images at 0.5, 4, and 24 hours after injection are shown in Figure 4. Labeled 17-AAG administered intratumorally had significantly high activity in H460 tumors and less distribution in normal organs. In contrast, the images acquired in the i.v. administration group showed physiologic, but intense, localization of ¹³¹I-17-AAG in the liver, spleen, kidneys, and urinary bladder at 0.5 hour postinjection. The distribution of radioligand in the tumor area appeared at 24 or 48 hours according to individual differences in mice, but was not as significant as in intratumoral administration. The excretion of Na¹³¹I through intratumoral injection appeared to be a nonspecific binding process mainly through the urinary system, without tumor retention. The in vivo uptake was comparable to background tissue activity at 48 hours postinjection. One (1)-week images (not shown here) demonstrated that only in the intratumoral injection group did the activity of the radiolabel appear in the tumor region. The 7-day uptake of ¹³¹I-17-AAG in the dissected tumors was 25.43 ± 7.37% ID/g in the intratumoral injection group, which was statistically significant (p < 0.05) compared with the uptake in the i.v. and control groups (0.73 ± 0.08 and 0.11 ± 0.04% ID/g, respectively). However, in the blocking study, in which mice were pretreated with unlabeled 17-AAG, the tumor uptake of ¹³¹I-17-AAG reduced to 39.56 ± 9.83% ID/g at 24 hours postinjection, which was significantly different from the 24-hour uptake (121.10 ± 31.63% ID/g) of the radiolabel alone applied intratumorally (p < 0.05).

FIG. 4.

In vivo SPECT images (shown in black and white) of H460 tumor-bearing mice at 30 minutes, 4 hours, and 24 hours after injection of 5.55 MBq radiotracers in 100 μL per mouse, using the same number of mice in each group. (A) ¹³¹I-17-AAG intratumoral injection group, (B) ¹³¹I-17-AAG tail vein injection group, (C) Na¹³¹I intratumoral injection group, (D) the blocking group preinjected with unlabeled 17-AAG. Arrows indicate the tumor site. 17-AAG, 17-allylamino-17-demethoxy-geldanamycin.

Cancer treatment analysis

Figure 5 shows the tumor-growth curves of different ¹³¹I-17-AAG treatment groups, compared with the natural tumor-growth curve of Group 7. The 16-day tumor volumes were 384.60 ± 113.74, 492.20 ± 61.34, 668.40 ± 197.76, 1044.30 ± 199.45, 1140.13 ± 77.04, 1455.65 ± 66.25, and 1781.20 ± 127.81 mm³, respectively, in Groups 1–7. All treatment groups had a degree of tumor-growth inhibition compared with the control group (p < 0.05). The tumor inhibition ratios of Groups 1–6 were 88.41% ± 3.69%, 76.57% ± 4.81%, 60.35% ± 5.47%, 38.50% ± 10.95%, 35.45% ± 7.85%, and 21.05% ± 4.33%, respectively (Fig. 6). Overall, tumor treatment effects in the intratumoral groups were higher than in the i.v. groups (p < 0.05). Through intratumoral administration, Group 1 administered 5.55 MBq of ¹³¹I-17-AAG twice at an 8-day interval obtained the optimal inhibitive ratios, even better than Group 2 at a single dose of 11 MBq (p < 0.05). However, no significant differences appeared in Groups 4 and 5 via i.v. administration (p > 0.05). Regardless of the administration route, the 11.0 MBq-dose groups produced greater efficacy than the 5.5 MBq-dose groups (p < 0.05).

FIG. 5.

Comparison of tumor volumes over 16 days in H460 tumor-bearing nude mice after different treatments, versus untreated mice. The 16-day tumor volumes were significantly different (F = 60.54; p < 0.01).

FIG. 6.

Tumor inhibition ratios at 16 days after treatment, in H460 tumor-bearing nude mice, compared with untreated group. There were statistical significances among groups (F = 61.19; p < 0.01).

Histologic correlation

Histopathologic examination revealed no evidence of treatment-induced abnormalities in the heart, liver, lungs, sternum, intestines, or kidneys of mice. Histological analysis of H&E-stained tumor tissues demonstrated that the control group presented with intact tumor cell structure (Fig. 7A), while tumors treated with ¹³¹I-17-AAG showed distinct damage of individual tumor cells (Fig. 7B). The results of IHC staining are shown in Figures 7B–F and 8. HSP90α antigens were mainly distributed in the cytoplasm, with a small amount of expression in the nuclei. The HSP90α expression correlated positively with tumor inhibition (r = 0.912, p < 0.01). Statistically, after 16-day ¹³¹I-17-AAG treatment with radioactivity 11.0 MBq, the percent of the HSP90α+ fraction was 84.38% ± 7.95% in the untreated group, which differed from 62.38% ± 8.16% in the i.v. and 26.25% ± 7.77% in the intratumoral administration groups (t = 3.86 and 10.45, respectively; p < 0.05). The proliferation index Ki-67 was 70.38% ± 4.85% in the control group, which was also statistically different from 52.63% ± 5.31% in the i.v. and 39.38% ± 4.78% in the intratumoral administration groups (t = 4.93 and 9.10, respectively, p < 0.05). A significant correlation existed between the HSP90α+ and Ki-67+ fractions (r = 0.878, p < 0.01).

FIG. 7.

Effects of H460 tumor cells were observed in the control group and the ¹³¹I-17-AAG treatment groups. H&E staining (shown in black and white) showed that cells in the control group grew in good condition, 400× magnification (A), and tumor tissue necrosis and cell structure damage were seen in the ¹³¹I-17-AAG treatment groups, 200× magnification (B). IHC staining showed HSP90α positivity in the control group (C) and relatively low positivity after ¹³¹I-17-AAG treatment (D), as well as Ki-67 positivity in the control group (E) and relatively low positivity after ¹³¹I-17-AAG treatment (F), 200× magnification. 17-AAG, 17-allylamino-17-demethoxygeldanamycin; H&E, hematoxylin and eosin; IHC, Immunohistochemical; HSP90, heat shock protein 90.

FIG. 8.

Comparison of HSP90α+ fractions and Ki-67+ fractions among the intratumoral injection group, the intravenous injection group, and the control group after IHC staining of tumor tissue sections. The H460 tumor-bearing mice were treated with radioactivity 11.0 MBq of ¹³¹I-17-AAG for 16 days using different treatments. HSP90, heat shock protein 90; IHC, immunohistochemical; 17-AAG, 17-allylamino-17-demethoxygeldanamycin.

Discussion

In this study, the combination of radionuclide ¹³¹I and 17-AAG was validated in living mouse xenograft models. Biodistribution studies indicated relatively high tumor uptake of radiolabeled ligands in these human NSCLC xenograft models, with very low distribution in normal organs at 24 hours postinjection. Intratumoral delivery improved the tumor uptake of labeled 17-AAG by increasing tumor concentration and decreasing the uptake in healthy organs. In vivo imaging also showed relatively high retention of radiolabels in tumor regions, especially with intratumoral injection. Competition study verified that ¹³¹I-17-AAG had a specific affinity for tumor tissues. Compared to nonspecific distribution in the Na¹³¹I control, 1-week radioactive retention within the tumor in the treatment groups demonstrated that the radiolabeled 17-AAG compound may have a certain in vivo biological stability and pharmacodynamic potential in the tumor sites.

All treatment groups effected tumor inhibition compared with the untreated group. The inhibitive efficacy of ¹³¹I-17-AAG injected intratumorally was better than that injected i.v. (p < 0.05). Tumor inhibition ratios for intratumoral administration were nearly two to three times higher than those for i.v. administration for the same dose of injection. Intratumoral injecting carrier-based radiotherapy could be an interesting option, as it may avoid whole body distribution and excretion and reduce the possible toxicity involved in the i.v. injection route. By both routes, the 11.0 MBq-dose groups were more efficient than the 5.5 MBq-dose groups. This finding may indicate a certain dose–effect relationship for ¹³¹I-17-AAG. The difference between single dose and fractionated dose was not significant and found only via intratumoral delivery. But repeated low-dose administration could still be considered in treatment. Regardless of the delivery method or dose, ¹³¹I-17-AAG undoubtedly has therapeutic effects on NSCLC tumor models, as is evident from this study.

Increased expression of HSP90 above the level observed in normal tissues is a common feature of most human cancers, including both solid tumors and hematological malignancies.² Braga-Basaria et al. reported using a thyroid cancer model that showed that sensitivity of cancer cells to 17-AAG-induced cytotoxicity relates to HSP90 levels rather than to histological subtype.²⁴ In addition to various client proteins, HSP90 itself and the cellular stress response seem to be important determinants of drug sensitivity.^25,26 The IHC results of the present study showed that the expression of HSP90α, the inducible and major form of HSP90,²⁷ correlated significantly with tumor treatment activity. The average HSP90α+ fraction was 26.01% in the optimal treatment groups given intratumoral injection, while it was 61.57% in the i.v. administration groups and 84.13% in the untreated group. Moreover, the proliferation index Ki-67, which is a valuable marker in the prognostication of tumor-cell proliferation activity,²⁸ also had a positive correlation with HSP90α expression (r = 0.878, p < 0.01). The results verify further that ¹³¹I-17-AAG could play an effective role in H460 NSCLC, by inhibiting HSP90α protein expression and achieving a target-specific role in lung-cancer treatment.

As mentioned in the Introduction, 17-AAG in patients with refractory cancer usually achieves only a protracted stabilization of disease, but requires relatively high doses and long schedules.^{9
–11,29,30} For example, Ramanathan et al. recommended phase II doses of 175 to 200 mg/m² of 17-AAG twice weekly,³¹ and Banerji et al. recommended i.v. administration at a dose of 450 mg/m² once weekly.^10,32 Moreover, preclinical data showed that geldanamycin achieved a 50% growth inhibition at 13 nM in highly responsive cell lines, with an overall mean sensitivity of 180 nM.³³ Gallegos Ruiz et al. reported a 50% growth inhibition by 17-AAG at around 30 nm against H460 NSCLC tumor cells.³⁴ In the present study, only about 37.5 pmol (5.55 MBq radioactivity) or 75.0 pmol (11.0 MBq radioactivity) of radiolabeled 17-AAG was used per mouse. The therapeutic effects of 17-AAG on tumors would be quite limited at these dosages. However, significant tumor inhibition was observed just after 1 week of therapy using the radiolabeled 17-AAG compound. Thus, radionuclide irradiation has definite therapeutic efficacy and retention role in tumor treatment. The results of in vivo studies suggest that a combination of a 17-AAG treatment program and labeled 17-AAG for radiotherapy may be effective for treating refractory and advanced solid malignancies, which may shorten the courses of 17-AAG treatment, decrease the amount of 17-AAG, and especially improve the efficacy of cancer treatment.

Furthermore, the limited water solubility of 17-AAG, the natural product of HSP90 inhibitors, may pave the way for the development of more effective and flexible formulation of inhibitors combined with radiotherapy. The selection and comparison of other radionuclides, for example, samarium-153, phosphorus-32, and yttrium-90, may be necessary for obtaining more effective therapeutic results.

Conclusions

Using radionuclide-labeled HSP90 inhibitors may be a considerable means of further tumor treatment.

Footnotes

Acknowledgments

This study was supported by the National Advanced Technique Investigation and Development Project of China (863 Project, No. 2007AA02Z471), the National Natural Science Fund of China (No. 30470500), and the Jiangsu Province scientific and technological innovation projects for graduate students (China, No. CX09B_068Z).

Disclosure Statement

No potential conflicts of interest were disclosed.

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