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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Medullary thyroid carcinoma is a rare endocrine tumor, which shows overexpression of somatostatin receptor subtype 2. There is no systemic therapy for medullary thyroid carcinoma. Previously we reported that octreotide-PEG liposomes loaded with irinotecan, which target somatostatin receptor subtype 2, showed high therapeutic efficacy for medullary thyroid carcinoma xenografts compared with free irinotecan or non-targeted non-PEGylated liposomal irinotecan. In this study, we evaluated octreotide-PEG liposomes loaded with irinotecan in terms of the biodistribution of irinotecan and its active metabolite, and its therapeutic efficacy, compared with PEGylated liposomes. Furthermore, to elucidate the effect of octreotide ligand after cellular association, we assessed the cytotoxicity in tumor cells and the inhibition of protein phosphorylation in the tumor cells and xenografts using empty octreotide-PEG liposomes, which were loaded with no drug. In a therapeutic study, octreotide-PEG liposomes loaded with irinotecan significantly improved median survival compared with PEGylated liposomes. In tumor tissue at 6 h after injection, octreotide-PEG liposome-treated mice showed significantly higher concentrations of irinotecan and 7-ethyl-10-hydrocamptothecin compared with PEGylated liposome-treated mice, indicating that octreotide-PEG liposomes accumulated rapidly and to a high level in the tumor. Furthermore, empty octreotide-PEG liposome inhibited the phosphorylation of p70S6K in vitro and in vivo. These findings indicated that octreotide-PEG liposomal irinotecan has dual functions with targeted tumor delivery and assistance of cellular cytotoxicity, which led to higher therapeutic efficacy than PEGylated liposomes for medullary thyroid carcinoma xenografts. (Cancer Sci 2012; 103: 310–316)

Medullary thyroid carcinoma (MTC) originates in C cells of the thyroid gland. It is known to overexpress somatostatin receptor (SSTR) subtypes 1, 2, and 5.(1) Somatostatin receptor 2 expression was the most frequently detected subtype in human MTC and was significantly higher than the other SSTR subtypes in an established human MTC cell line, TT.(1–3) A therapeutic approach for MTC using irinotecan (CPT-11) was reported, but the results were inconclusive.(4,5) Overall, there is no systemic therapy for MTC.

CPT-11 is a water-soluble prodrug and is converted to 7-ethyl-10-hydrocamptothecin (SN-38), an active metabolite of CPT-11, by carboxylesterase (CE).(6,7) CPT-11 inhibits the resealing of single-strand DNA breaks mediated by topoisomerase I by stabilizing cleavable complexes and is a cell cycle-specific drug.(8–11) From this, a long period of exposure to CPT-11 induces cytotoxicity of tumor cells. The CPT-11 therapy, however, has failed owing to its short half-life and low tumor distribution. Therefore, selective delivery of CPT-11 to tumor sites could lead to successful CPT-11 therapy for MTC. For this purpose, we developed Oct-CL, namely, octreotide-PEG liposomes loaded with CPT-11, in which octreotide (Oct) has high binding affinity for SSTR2.(12) This approach proved promising because Oct-CL led to higher therapeutic efficacy for MTC xenografts than free CPT-11 or non-targeted non-PEGylated liposomal CPT-11.(13)

PEGylated liposomes (SL) can passively accumulate into tumor tissue due to the enhanced permeability and retention effect.(14) Therefore, we compared the therapeutic efficacy of antitumor activity of Oct-CL with SL to clarify whether the active-targeting ability of Oct-CL was superior to the passive-targeting ability of SL. Generally, because active-targeting liposome biodistribution is different from that of passive-targeting liposomes, we examined the biodistribution of CPT-11 and SN-38 after i.v. injection of Oct-CL and SL into MTC xenograft mice.

Moreover, we found that non-loaded Oct-CL, or empty Oct-CL, showed higher cytotoxicity in TT cells compared with empty SL.(13) To gain more insight into tumor suppression, we examined the mechanism of cytotoxicity of the Oct ligand. It was reported that Oct alone can produce an antiproliferative action to inhibit the phosphorylation of p70S6K in the Akt/mTOR/p70S6K pathway in insulinoma cells.(15) From this information, we tried to clarify the effects of Oct ligand on the phosphorylation of p70S6K in vitro and in vivo. We provide an answer to the mechanism of cytotoxicity of empty Oct-CL in this study.

Here we present a more extensive investigation of Oct-CL. We evaluated the function of Oct using Oct-CL from viewpoints of therapeutic efficacy and the biodistribution of CPT-11 and SN-38 in MTC tumor xenografts after injection of Oct-CL or SL. Furthermore, we assessed the cytotoxicity mechanism of Oct ligand using empty Oct-CL in vitro and in vivo.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Materials.  CPT-11 was a kind gift from Yakult (Tokyo, Japan). Distearoylphosphatidylcholine (DSPC) and methoxy-PEG2000-distearoylphosphatidylethanolamine (PEG-DSPE) were purchased from NOF (Tokyo, Japan). Oct-PEG3400-DSPE(16) was purchased from KNC Laboratories (Kobe, Japan). SN-38 and cholesterol were purchased from Wako Pure Chemical Industries (Osaka, Japan). Phytic acid (IP-6) solution was obtained from Nacalai Tesque (Kyoto, Japan). Other reagents were of analytical or HPLC grade.

Preparation of liposomal CPT-11.  Liposomal IP6 was prepared from DSPC/cholesterol at a molar ratio of 55/45, as reported previously.(13) Extra IP6 solution of liposomal IP6 was then exchanged for HBS-buffer (20 mM HEPES, 150 mM NaCl, pH 7.4) using a Sephadex G-50 column. The liposomal IP6 was loaded with CPT-11 aqueous solution at 60°C for 1 h. It was then incubated with 1.6 mol% Oct-PEG-DSPE for Oct-CL or 1.6 mol% PEG-DSPE for SL at 60°C for 25 min.(13) Empty liposomes were prepared using the same protocol but without loading drug. The particle size and zeta-potential were measured using an ELS-Z2 (Otsuka Electronics, Osaka, Japan) at 25 ± 1°C after diluting the liposome suspension in water.

Cytotoxicity assay on TT cells.  Cytotoxicity on TT cells (human MTC cell line) was carried out using free Oct or empty liposomes. The Oct concentration of empty Oct-CL was measured by an Oct-EIA kit (Peninsula Laboratories, LLC, San Carlos, CA, USA). Cells were incubated for 48 h at the different concentrations of free Oct, empty liposomes in the presence or absence of 1.5 μM CPT-11, which corresponded to one-tenth of the 50% growth-inhibitory concentration.(13) The lipid amount of empty SL corresponded to that of empty Oct-CL. Cell viability was measured by a cell proliferation kit (Dojindo, Kumamoto, Japan) as reported previously.(13)

Animals.  All animal experiments were carried out with approval from the Institutional Animal Care and Use Committee at Hoshi University (Tokyo, Japan). TT cells (1 × 107) were inoculated s.c. into female ICR nu/nu mice (6 weeks of age; Oriental Yeast, Tokyo, Japan).

Antitumor effects.  When the average tumor volume was approximately 100 mm3, Oct-CL or SL was injected i.v. at a dose of 10 mg CPT-11/kg in two injections at 3-day intervals (23.9 mg lipid/kg/injection). Empty Oct-CL or empty SL was injected i.v. (23.5 mg lipid and 867 nmol Oct/kg/injection, corresponding to Oct ligand amount for the therapeutic study) in two injections at 3-day intervals. Tumor volume and body weight were measured for individual animals. Tumor volume was calculated using the following equation: volume = 1/2ab2, where a is the long diameter and b is a short diameter.

Biodistribution in TT xenograft mice by HPLC.  When the average tumor size reached approximately 100 mm3, mice were treated with Oct-CL or SL i.v. at a dose of 10 mg CPT-11/kg. At 6 h and 24 h after a single injection, blood was collected. Tumor, liver, kidneys, lung, and spleen were excised and homogenized. CPT-11 and SN-38 were extracted using ice-cold acidic methanol and analyzed by HPLC methods, as reported previously.(17) The CPT-11 dose %/mL plasma or g tissue was calculated as the amount of CPT-11 per total plasma volume (mL) or per total tissue weight (g), respectively, from that of injected liposomal CPT-11.(17)

Distribution of liposomal CPT-11 in TT xenograft mice by fluorescence microscope.  When the average tumor size reached approximately 150 mm3, mice were treated with Oct-CL or SL i.v. at a dose of 10 mg CPT-11/kg. At 6 and 24 h after a single injection, tissues were collected and prepared as 20-μm frozen sections. Tissue sections were examined using an inverted microscope, ECRIPS TS100 (Nikon, Tokyo, Japan), with an Epi-Fluorescence Attachment (Nikon) using a UV1A filter.

Conversion activity of CPT-11 by CE.  The conversion activity of CPT-11 by CE (CPT-CE activity) in TT cells, normal liver, and TT tumor tissue was measured by the Guichard method.(18) Briefly, TT cells (4 × 104 cells) were homogenized. The homogenates were centrifuged at 20 000 g for 30 min at 4°C to obtain cytosol. Cytosolic protein (3 mg/mL, 80 μL) and 5 μM CPT-11 (20 μL) were mixed and incubated at 37°C for 1 h. Then ice-cold acidic acetonitrile was added to stop the reaction. SN-38 produced during the incubation was measured by an HPLC method, as previously reported.(17) The TT tumor tissue and liver were excised and homogenized. Cytosol from the TT tumor tissue and normal liver, which was perfused with ice-cold saline to remove blood, was prepared using the same protocol as for the extraction protocol in TT cells. CPT-CE activity in normal liver and TT tumor tissue was measured using the same protocol as for the assay protocol in TT cells.

Effects of empty liposomes, CPT-11, or SN-38 on the PI3K/Akt/mTOR/p70S6K pathway in TT cells.  TT cells were incubated with serum-starved (0.1% FBS) cell medium with empty Oct-CL, empty SL, CPT-11, or SN-38 for 24 h at 37°C. Cell lysates were prepared with ice-cold RIPA buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100 containing protease and phosphatase inhibitors), separated by SDS-PAGE, and blotted using standard procedures.(19) Primary antibodies were against Akt, tuberous sclerosis complex 2, tuberin (TSC2), p70S6K, phosphorylated Akt (Ser473), phosphorylated TSC2 (Thr1462), and phosphorylated p70S6K (Thr389) (all made in rabbits; Cell Signaling Technology, Beverly, MA, USA). Horseradish peroxidase-conjugated secondary antibody was used to detect the primary rabbit antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). All proteins were detected by peroxidase-induced chemiluminesence (Super Signal West Pico Chemiluminescent substrate; Pierce, Rockford, IL, USA).

Effects of empty liposomes on the PI3K/Akt/mTOR/p70S6K pathway in TT tumors.  When the average tumor size reached approximately 150 mm3, empty Oct-CL or empty SL was injected i.v. once (23.5 mg lipid and 867 nmol Oct/kg). At 24 h after injection, tumor tissue was collected and homogenized in RIPA buffer. Western blot analysis was carried out using the same protocol as for the in vitro study.

Statistical analysis.  The statistical significance of differences in the data was evaluated by analysis using one-way anova in combination with the Tukey–Kramer test. *P < 0.05 and **P < 0.01 were considered significant. Kaplan–Meier analysis was carried out using GraphPad Prism version 4.0 (GraphPad Software, San Diego, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Size and zeta-potential of liposomes.  Two types of liposomes, 1.6 mol% Oct-PEG-DSPE modified Oct-CL and 1.6 mol% PEG-DSPE modified SL were prepared (Table 1), because we reported previously that 1.6 mol% Oct modification was effective for association with TT cells.(13) The average particle size of each liposome was ∼146 nm with a narrow monodisperse distribution. The zeta-potential and drug entrapment efficiency of each liposome formulation was approximately −14 mV and >80%, respectively. There were no significant differences between Oct-CL and SL in terms of particle characters such as particle size, zeta-potential, or drug entrapment efficiency, except for active-targeting ability.

Table 1.   Particle size and zeta-potential of liposomal CPT-11
FormulationSize (nm)Zeta-potential (mV)
  1. Values represent the mean ± SD (n = 3). Oct-CL, non-PEGylated liposomes modified with 1.6 mol% octreotide-methoxy-PEG3400-distearoylphosphatidylethanolamine; SL, PEGylated liposomes modified with 1.6 mol% methoxy-PEG2000-distearoylphosphatidylethanolamine.

Oct-CL146.9 ± 20.1–14.0 ± 1.3
SL146.2 ± 15.0–13.4 ± 0.9

Therapeutic efficacy of liposomal CPT-11.  The antitumor effect of Oct-CL and SL was evaluated in TT tumor xenografts. Oct-CL showed significantly stronger antitumor effects at days 7–25 after the treatment compared with saline and at days 7–15 compared with SL (Fig. 1a). Body weight losses were not observed (Fig. 1b). Oct-CL treatment significantly improved median survival up to 212 days, compared with 198 and 121 days observed in the SL-treated group (P < 0.01) and saline-treated group (P < 0.05), respectively (Fig. 1c).

image

Figure 1.  Therapeutic efficacy of treatment for medullary thyroid carcinoma using octreotide-PEG liposomes (Oct-CL) or PEGylated liposomes (SL). Mice were injected i.v. with Oct-CL (○), SL (▪) (equivalent to 10 mg CPT-11/kg), or saline (Δ), as shown by arrows. Their treatment effects on tumor size (a), body weight (b), and survival rate (c) were measured. Each value represents the mean ± SD (n = 6). *P < 0.05 versus Oct-CL-treated mice.

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Biodistribution of liposomal CPT-11.  The biodistribution of CPT-11 and SN-38 was examined in TT xenografts at 6 or 24 h after i.v. injections of Oct-CL or SL loaded with CPT-11. CPT-11 was highly distributed in the liver and spleen (Fig. 2). Frozen sections of tumors in the Oct-CL treated group were observed with blue fluorescence of CPT-11 than those in SL treated group, both 6 and 24 h after injection (Fig. 2a). SN-38 was not able to be detected under this condition. CPT-11 blue fluorescence was weak and was not enough to show significant difference in CPT-11 tumor accumulation between Oct-CL- and SL-treated groups. Therefore, we next measured concentrations of CPT-11 and SN-38 in tumor tissues and other organs by the HPLC method. Six hours after injection, CPT-11 levels in the liver of Oct-CL-treated mice were significantly higher than that of SL-treated mice (P < 0.05) (Fig. 2b). CPT-11 and SN-38 levels in tumor tissue of Oct-CL-treated mice were significantly higher, 3.7- and 2-fold, respectively, compared with that of SL-treated mice (P < 0.01) (Fig. 2b,c). However, the kidney distribution of CPT-11 in Oct-CL-treated mice was significantly lower than that of SL-treated mice (P < 0.01). Twenty-four hours after injection, SL-treated mice maintained a higher CPT-11 level in the plasma (7.1 ± 2.4% dose/mL), whereas Oct-CL-treated mice showed approximately half the CPT-11 plasma concentration of SL-treated mice (P < 0.05). Tumor CPT-11 concentration between the Oct-CL- and SL-treated groups was not significantly different, but the SN-38 concentration in the Oct-CL-treated group was significantly higher, 2.2-fold, than in SL-treated mice (P < 0.01).

image

Figure 2.  Biodistribution of liposomal CPT-11 (Oct-CL) at 6 or 24 h after a single injection into mice. Frozen sections of tumors were observed using a fluorescence microscope (a), and tissue biodistribution of CPT-11 (b) and SN-38 (c). Each value represents the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus PEGylated liposomes (SL). Blue fluorescence indicates CPT-11. Scale bars = 100 μm.

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In other organs, the results of the biodistribution of CPT-11 measured by HPLC agreed with the observation of blue fluorescence of CPT-11 (Fig. S1).

In vitro conversion of CPT-11 to SN-38 in TT cells, liver, and TT tumor tissues.  In the biodistribution study, higher SN-38 accumulation was observed in tumor tissues 6 and 24 h after injection of Oct-CL compared to SL. To confirm the conversion of CPT-11 to SN-38 in TT cells, we analyzed the in vitro conversion of CPT-11 to SN-38 in TT cells. The cytosol of TT cells showed the conversion of CPT-11 to SN-38 at a rate of 2.53 ± 0.09 pmol/h/mg protein (data not shown). TT cells per se have the conversion activity of CPT-11 to SN-38. With regard to TT tumor tissue, the conversion rate in the liver was approximately 8.5% of the CPT-11 that was initially added to the reaction mixture (100 pmol CPT-11) (Fig. 3). Interestingly, the conversion of CPT-11 to SN-38 activity in TT tumor tissue was 0.8-fold that of the liver, suggesting that CPT-11 loaded into Oct-CL was accumulated in the tumor directly by active targeting of Oct-CL, and was converted into SN-38. The negative control (initially adding the stop solution before the addition of CPT-11 and incubation) showed no production of SN-38.

image

Figure 3.  CPT-11 converted by carboxylesterase (CE) activity. Cytosol was incubated with CPT-11 at 37°C for 1 h. Each value represents the mean ± SD (n = 3). *P < 0.05.

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Effects of Oct ligand of liposomes on growth inhibition and phosphorylation of p70S6K.  As shown in Figure 4(a), empty Oct-CL exerted a growth inhibitory effect on TT cells in a dose-dependent fashion of Oct ligand. Furthermore, addition of 1.5 μM CPT-11 potentiated the growth inhibition activity of empty Oct-CL, having led to an additional ∼20% reduction in cell viability. In contrast, empty SL did not exert any growth inhibitory effect in the presence or absence of CPT-11 (Fig. 4b). Free Oct showed a growth inhibitory effect only at high concentration, 100 μM (Fig. 4c), but showed no synergistic effect with CPT-11.

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Figure 4.  Cytotoxicity of free octreotide (Oct) or empty liposomes on human medullary thyroid carcinoma TT cells in the presence or absence of CPT-11. The TT cells were treated with empty octreotide-PEG liposomes (Oct-CL) (a), empty PEGylated liposomes (SL) (b), or free Oct (c), alone or combined with CPT-11. Each value represents the mean ± SD (n = 4). DSPE, distearoylphosphatidylethanolamine. *P < 0.05 versus control (cell viability of non-treatment); #P < 0.05 versus CPT-11 alone.

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In many cancers, the PI3K/Akt/mTOR pathway contributes to cell proliferation and growth, and this pathway is activated by phosphorylation of Akt, TSC2, or p70S6K proteins.(12,20–25) Therefore, we tried to clarify the effects of Oct ligand on phosphorylation of proteins in TT cells and TT tumors by Western blotting, using empty Oct-CL, empty SL, CPT-11, or SN-38. As shown in Figure 5(a), empty SL and empty Oct-CL did not affect the phosphorylation of either Akt or TSC2 in TT cells. Only empty Oct-CL (corresponding to 0.42 μM Oct ligand) strongly inhibited the phosphorylation of p70S6K at the Thr389 site, whereas total p70S6K was not affected. In TT tumor tissue, empty Oct-CL or empty SL did not affect total protein p70S6K. Mice injected with empty Oct-CL showed a decrease in the level of phosphorylated p70S6K, but those with empty SL did not (Fig. 5b). However, the empty Oct-CL and the empty SL did not show antitumor effects under this experiment condition in TT tumor xenografts (Fig. 6).

image

Figure 5.  Effects of empty PEGylated liposomes (SL) or empty octreotide-PEG liposomes (Oct-CL) on Akt-TSC2-p70S6K in human medullary thyroid carcinoma TT cells (a) and TT tumor tissue (b). TT cells were treated with empty Oct-CL (corresponding to 0.42 μM Oct-PEG-distearoylphosphatidylethanolamine [DSPE]), empty SL liposomes modified with 0.42 μM PEG-DSPE, CPT-11 (13 μM), or SN-38 (0.14 μM) for 24 h. TT tumor-bearing mice were injected with empty Oct-CL 24 h before the experiment. UNT, untreated.

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image

Figure 6.  Therapeutic efficacy of injection of empty octreotide-PEG liposomes (Oct-CL) or empty PEGylated liposomes (SL) into mice bearing human medullary thyroid tumors at a dose of 23.5 mg lipid and 867 nmol Oct/kg/injection or 23.5 mg lipid, respectively. Treatment effects of empty liposomes on tumor size (a) and body weight (b) were measured. The formulations used were empty Oct-CL (○), empty SL (▪), and saline (Δ). Each value represents the mean ± SD (n = 6). Arrows indicated the day of drug injection.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

In this study, we showed that Oct-CL loaded with CPT-11 showed the early and higher accumulation of CPT-11 in tumor, enhanced the antitumor effect, and significantly prolonged median survival compared with SL. Moreover, the mechanism of action of Oct associated with liposomes was investigated by measuring the biodistribution of Oct-CL loaded with CPT-11 and the phosphorylation of proteins after empty Oct-CL treatment. Recently, we have reported that Oct-CL loaded with CPT-11 showed higher therapeutic efficacy compared with free CPT-11 or non-targeted non-PEGylated liposome in TT tumor xenografts.(13) However, the effect of Oct and its mechanism still remained after association with liposomes.

In our study, Oct-CL showed a significantly higher distribution to TT tumor tissue compared with SL at least until 6 h after injection. This result suggested that Oct-CL accumulated rapidly in the tumor after injection as a result of Oct-targeting, whereas SL accumulated slowly in the tumor by the enhanced permeability and retention effect for 24 h. Twenty-four hours after injection, there was no significant difference between Oct-CL and SL. The CPT-11 level of Oct-CL in the tumor was sustained for 24 h after injection.

Why was the SN-38 concentration of tumors in Oct-CL-treated mice significantly higher than that of SL-treated mice at 24 h even though the CPT-11 concentration was similar? Generally, it is reported that CPT-11 conversion to SN-38 mainly occurs through the action of liver CE.(26) Accordingly, it can be regarded that SN-38 transformed in the liver accumulated in the tumor. When the accumulation of CPT-11 in the liver was similar between Oct-CL- and SL-treated mice, converted SN-38 in the liver should be present in similar amounts, and, therefore, the amount of SN-38 accumulating in tumor should be similar. We previously reported that there were no significant differences between Oct-CL- and SL-treated mice in terms of drug release over 48 h at 37°C in PBS.(13) Furthermore, an in vitro conversion study showed that the conversion activity of CPT-11 to SN-38 in TT tumor tissue was 0.8-fold that of the liver. From these findings, higher SN-38 concentration of Oct-CL at 24 h might reflect early direct distribution of Oct-CL to tumor tissue by SSTR targeting. To the best of our knowledge, this is the first report regarding CPT-11 conversion activity in TT cells and TT tumor tissue.

When we examined other tissues, there were significant differences between Oct-CL- and SL-treated mice in terms of kidney and liver distribution. The CPT-11 concentration in the kidneys of Oct-CL-treated mice was significantly lower compared with that of SL-treated mice. Because the kidney is known to express SSTR2,(27) Oct-CL might be selectively and rapidly distributed to the kidneys, as well as to the tumor, then excreted more rapidly than in SL-treated mice. With regard to the liver, Oct-CL accumulated to a higher level in the liver than SL, because SL was modified with Oct ligand and it may be taken up by the reticuloendothelial system.

Empty Oct-CL exerted cell growth inhibition at low concentrations (0.42 and 4.2 μM), whereas free Oct did not exert cell growth inhibition below 100 μM (Fig. 4a,c). Empty Oct-CL showed effective cell growth inhibition compared with free Oct, suggesting that the affinity of empty Oct-CL to SSTR may be higher than that of free Oct. Phosphorylation of p70S6K is reported to activate cell growth and proliferation.(24) p70S6K is the downstream protein of the PI3K/Akt/mTOR pathway and phosphorylation of p70S6K is generally used as a marker of the inhibition of the PI3K/Akt/mTOR pathway.(28) Treatment with empty Oct-CL caused the suppression of phosphorylation of p70S6K in vitro and in vivo (Fig. 5a,b) but empty SL did not. Therefore, this result suggested that empty Oct-CL inhibited cell growth and proliferation by suppressing the phosphorylation of p70S6K in TT cells and TT tumor tissue. In other words, the targeting ligand Oct had not only tumor targeting activity but also assisted with antitumor effects. Therefore, the Oct ligand has dual functionality.

However, treatment with empty Oct-CL did not show any antitumor effect under this condition (Fig. 6). For MTC, it was reported that clinically s.c. injection of free Oct at 500 μg (=458 nmol)/day for 90 days, and 150 μg (=137 nmol)/day for 6 months showed no effect on therapeutic efficacy.(29,30) In this therapeutic study, Oct originated from Oct-CL was injected twice as 867 nmol Oct/kg/day. From these different schedules, it is difficult to judge the therapeutic efficacy by Oct alone in our study.

Figure 7 illustrates the proposed antitumor effects of Oct-CL loaded with CPT-11 for MTC. Oct-CL was selectively associated with TT cells. CPT-11 was released from Oct-CL, then CPT-11 was converted to SN-38 by CE in TT tumor. SN-38 produced in the tumors showed cytotoxicity. Oct-CL suppressed the phosphorylation of p70S6K. These suppressions led to inhibition of cell growth and proliferation, which assisted in antitumor effects for MTC using Oct-CL loaded with CPT-11.

image

Figure 7.  Proposed antitumor effects of octreotide-PEG liposomes loaded with CPT-11 (Oct-CL) for human medullary thyroid carcinoma.

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In conclusion, the present study showed that Oct-CL loaded with CPT-11 showed enhanced therapeutic efficacy, which correlated with a strong antitumor effect and the significant improvement of median survival, which was superior to SL in TT tumor xenografts. The improvement in therapeutic efficacy was due to the dual functions of Oct; the rapid and highly selective distribution of Oct-CL to tumor by SSTR targeting and the assistance of cell growth suppression by Oct.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology of Japan and by the Open Research Center Project.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. Disclosure Statement
  8. References
  9. Supporting Information

Fig. S1. Distribution of liposomal CPT-11 in the liver, spleen, lung, and kidneys.

FilenameFormatSizeDescription
CAS_2128_sm_fS1.jpg31KSupporting info item

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