Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro
Aim: To evaluate the effects of paclitaxel-octreotide conjugates on the growth of cultured non-small cell lung cancer cells. Methods: RT-PCR was performed to detect mRNA for the subtypes of the human somatostatin receptor (SSTR) using specific primers. MTT-based cytotoxicity assay was used to evalua...
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Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України
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| Цитувати: | Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro / M.L. Sun, J.M. Wei, X.W. Wang, L. Li, P. Wang, M. Li, C.H. Yi // Experimental Oncology. — 2007. — Т. 29, № 3. — С. 186–191. — Бібліогр.: 32 назв. — англ. |
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irk-123456789-1389592018-06-20T03:06:04Z Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro Sun, M.L. Wei, J.M. Wang, X.M. Li, L. Wang, P. Li, M. Yi, C.H. Original contributions Aim: To evaluate the effects of paclitaxel-octreotide conjugates on the growth of cultured non-small cell lung cancer cells. Methods: RT-PCR was performed to detect mRNA for the subtypes of the human somatostatin receptor (SSTR) using specific primers. MTT-based cytotoxicity assay was used to evaluate the cell viability after treatment with paclitaxel and the conjugates. Cell cycle perturbations were determined using a Fluorescence-Activated Cell Sorter. Results: Non-small cell lung cancer A549 and Calu-6 cells expressed mRNA for SSTR2 and SSTR5. Paclitaxel and the conjugates effectively inhibited the growth of A549 and Calu-6 cells in a concentration- and time-dependent manner. In SSTR-negative fibroblasts, the conjugates were less cytotoxic than paclitaxel. The conjugates and paclitaxel could induce the increase of G2/M phase ratio in A549 cells. Conclusion: The paclitaxel-octreotide conjugates can be used as selective-targeted chemotherapeutic agents for treating non-small cell lung cancer. Цель: оценить эффект конъюгатов паклитаксела-октреотида на рост культивированных клеток немелкоклеточного рака легкого человека. Методы: для определения мРНК подтипов рецептора соматостатина человека (SSTR) применяли ОT-ПЦР. Анализ цитотоксичности в МТТ-тесте применяли для оценки выживаемости клеток после их инкубации с паклитакселом и конъюгатами. Нарушения клеточного цикла определяли с применением FACS — клеточного сортера. Результаты: установлено, что клеточные линии немелкоклеточного рака легкого A549 и Calu-6 экспрессируют SSTR2 и SSTR5 мРНК. Отмечено эффективное дозо- и времязависимое угнетение роста клеток A549 и Calu-6 паклитакселом и конъюгатами. Для SSTR-негативных фибробластов конъюгаты менее цитотоксичны, чем паклитаксел. Конъюгаты и паклитаксел могут индуцировать повышение соотношения фаз G2 /M в клетках A549. Выводы: конъюгаты паклитаксел-октреотида могут быть использованы как селективные химиотерапевтические агенты для воздействия на немелкоклеточный рак легкого. 2007 Article Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro / M.L. Sun, J.M. Wei, X.W. Wang, L. Li, P. Wang, M. Li, C.H. Yi // Experimental Oncology. — 2007. — Т. 29, № 3. — С. 186–191. — Бібліогр.: 32 назв. — англ. 1812-9269 http://dspace.nbuv.gov.ua/handle/123456789/138959 en Experimental Oncology Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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DSpace DC |
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English |
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Original contributions Original contributions |
| spellingShingle |
Original contributions Original contributions Sun, M.L. Wei, J.M. Wang, X.M. Li, L. Wang, P. Li, M. Yi, C.H. Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro Experimental Oncology |
| description |
Aim: To evaluate the effects of paclitaxel-octreotide conjugates on the growth of cultured non-small cell lung cancer cells. Methods: RT-PCR was performed to detect mRNA for the subtypes of the human somatostatin receptor (SSTR) using specific primers. MTT-based cytotoxicity assay was used to evaluate the cell viability after treatment with paclitaxel and the conjugates. Cell cycle perturbations were determined using a Fluorescence-Activated Cell Sorter. Results: Non-small cell lung cancer A549 and Calu-6 cells expressed mRNA for SSTR2 and SSTR5. Paclitaxel and the conjugates effectively inhibited the growth of A549 and Calu-6 cells in a concentration- and time-dependent manner. In SSTR-negative fibroblasts, the conjugates were less cytotoxic than paclitaxel. The conjugates and paclitaxel could induce the increase of G2/M phase ratio in A549 cells. Conclusion: The paclitaxel-octreotide conjugates can be used as selective-targeted chemotherapeutic agents for treating non-small cell lung cancer. |
| format |
Article |
| author |
Sun, M.L. Wei, J.M. Wang, X.M. Li, L. Wang, P. Li, M. Yi, C.H. |
| author_facet |
Sun, M.L. Wei, J.M. Wang, X.M. Li, L. Wang, P. Li, M. Yi, C.H. |
| author_sort |
Sun, M.L. |
| title |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| title_short |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| title_full |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| title_fullStr |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| title_full_unstemmed |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| title_sort |
paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro |
| publisher |
Інститут експериментальної патології, онкології і радіобіології ім. Р.Є. Кавецького НАН України |
| publishDate |
2007 |
| topic_facet |
Original contributions |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/138959 |
| citation_txt |
Paclitaxel-octreotide conjugates inhibit growth of human non-small cell lung cancer cells in vitro / M.L. Sun, J.M. Wei, X.W. Wang, L. Li, P. Wang, M. Li, C.H. Yi // Experimental Oncology. — 2007. — Т. 29, № 3. — С. 186–191. — Бібліогр.: 32 назв. — англ. |
| series |
Experimental Oncology |
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2025-07-10T06:55:48Z |
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2025-07-10T06:55:48Z |
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1837242050340192256 |
| fulltext |
186 Experimental Oncology 29, 186–191, 2007 (September)
Death from lung cancer is one of the most com-
mon types of cancer-related death. Non-small-cell
lung cancer (NSCLC) accounts for approximately 80%
of lung tumors. All the currently available treatment
options for NSCLC, including surgery, radiation, and
chemotherapy, have limited efficacy [1]. Surgery is
not an option for treatment because approximately
65% of these patients have advanced-stage (IIIB/IV)
disease at diagnosis [1]. In addition, NSCLC is more
resistant to chemotherapy than other forms of can-
cer [2]. Although some progress has been made
in the management of patients with NSCLC, there
is still a need to develop more efficacious methods
of treatment. Targeted chemotherapy is a modern
approach for the treatment of cancers because it is
more efficacious and less toxic than usual systemic
chemotherapy [3].
Somatostatin (SST) is a peptide hormone, the
cellular actions of which are mediated by a family of
G-protein-coupled receptors (SSTR) that has five sub-
types: SSTR1, SSTR2, SSTR3, SSTR4 and SSTR5 [4].
Some investigators have reported that cells of human
small cell lung cancer and NSCLC overexpress SSTRs
[5, 6]. The precise nature of the expression of SSTRs
by NSCLC cell lines remains controversial. Ferone et
al [7] detected SSTRs in Calu-6 cells, although nume-
rous investigators have reported that many NSCLC cell
lines do not express SSTRs [8, 9]. SST analogs may
be potential vehicles for chemotherapeutic drugs used
to treat SSTR-positive tumors because the binding of
SST to its receptor is followed by its internalization.
Therefore, the binding of an SST conjugate to cance-
rous cells that express SSTRs could result in the accu-
mulation of the chemotherapeutic drug malignant cells
[10]. Although this putative mechanism of action has
not yet been demonstrated for SST analogs carrying
cytotoxic drugs, the localization of primary tumors and
their metastases by scintigraphy using radiolabeled
SST analogs lend strong support for such a of this
mechanism of action [10, 11].
Paclitaxel is a diterpenoid taxane derivative that
was first isolated from Taxus brevifolia by Wani in 1971
[12]. It possesses excellent antitumor activity in a wide
variety of tumor models, especially in NSCLC [13]. Its
antitumor actions are attributed to its ability to promote
tubulin assembly into microtubules [14]. In the pres-
ence of paclitaxel, depolymerization of the microtu-
bules is inhibited, thereby interfering with the G2 and
M phases of the cell cycle [15]. In spite of its excellent
antitumor activity, paclitaxel is not cell specific.
Octreotide (SMS 201-995, SandostatinTM) is an
octapeptide analog of endogenous SST and binds to
SSTR-2, SSTR-3 and SSTR-5 [16]. By virtue of these
properties, we thought that octreotide had the poten-
tial be a suitable vehicle for delivering paclitaxel to its
intracellular target. Therefore, we developed a series
of cytotoxic octreotide conjugates that contain pa-
clitaxel and evaluated their cytotoxicity in two NSCLC
cell types, namely, Calu-6 cells, which are known to
express SSTRs and A549 cells, whose expression of
SSTRs is considered to be low [17].
MATERIALS AND METHODS
Materials. The human NSCLC A549 cell line was
obtained from the Institute of Biochemistry and Cell
Biology, Shanghai Institutes for Biological Sciences,
Chinese Academy of Sciences, Shanghai. The human
NSCLC cell line Calu-6 was purchased from Tianjin
PAcLITAxEL-OcTREOTIDE cONjugATES INHIbIT gROwTH
Of HuMAN NON-SMALL cELL LuNg cANcER cELLS in vitro
M.-L. Sun1, 2, J.-M. Wei1, X.-W. Wang1, *, L. Li3, P. Wang3, *, M. Li1, C.-H. Yi1
1Department of Oncology, Qilu Hospital, Shandong University, Jinan, 250012, China
2Central hospital of Jinan, Jinan, 250013, China
3School of Pharmaceutical Science, Shandong University, Jinan, 250012, China
Aim: To evaluate the effects of paclitaxel-octreotide conjugates on the growth of cultured non-small cell lung cancer cells. Methods:
RT-PCR was performed to detect mRNA for the subtypes of the human somatostatin receptor (SSTR) using specific primers.
MTT-based cytotoxicity assay was used to evaluate the cell viability after treatment with paclitaxel and the conjugates. Cell cycle
perturbations were determined using a Fluorescence-Activated Cell Sorter. Results: Non-small cell lung cancer A549 and Calu-6
cells expressed mRNA for SSTR2 and SSTR5. Paclitaxel and the conjugates effectively inhibited the growth of A549 and Calu-6 cells
in a concentration- and time-dependent manner. In SSTR-negative fibroblasts, the conjugates were less cytotoxic than paclitaxel.
The conjugates and paclitaxel could induce the increase of G2/M phase ratio in A549 cells. Conclusion: The paclitaxel-octreotide
conjugates can be used as selective-targeted chemotherapeutic agents for treating non-small cell lung cancer.
Key Words: non-small cell lung cancer, paclitaxel, octreotide.
Received: May 31, 2007.
Mei-li SUN and Jun-min WEI contributed equally.
*Corresponence: Fax: +86 (0) 531 86908010
E-mail: wangxw12@yahoo.com
*Fax: +86 (0) 531 88382731
E-mail: puwang@sdu.edu.cn
Abbreviations used: DEPC – diethylpyrocarbonate, HPLC – high-
performance liquid chromatography, NSCLC – non-small-cell
lung cancer, PyBOP – benzotriazol-1-yloxy-tris-pyrrolidinophos-
phonium, SST – somatostatin, SSTR – somatostatin receptor,
WP-06-1 – 2’-succinyl-paclitaxel.
Exp Oncol 2007
29, 3, 186–191
Experimental Oncology 29, 186–191, 2007 (September) 18729, 186–191, 2007 (September) 187September) 187) 187 187
Medical University Cancer Institute (China). Cultured
human fibroblast cells were obtained from human scar
tissue and were supplied by Dr. Bin Xu, of the Central
Laboratory of Qilu Hospital (China); and Dr. Bin Xu had
got the permission of Ethical committee and patients’
agreement for the cultured human fibroblast cells.
Roswell Park Memorial Institute Medium 1640 (RPMI),
fetal bovine serum, and typsin were purchased from
Invitrogen; TRIzol reagent and diethylpyrocarbonate
(DEPC) were purchased from Sangon, Shanghai;
RT-PCR Kit and DNA Marker DL2000 were purchased
from TaKaRa Biotechnology Co. Ltd. (China); The
five SSTR subtypes and β-actin primers were made
by Invitrogen. Paclitaxel was purchased from Man-
fangyuan Chemical Industry Co., Ltd., China; Octreo-
tide was purchased from Zillion Co. Ltd. (China); and
benzotriazol-1-yloxy-tris-pyrrolidinophosphonium
(PyBOP) was purchased from Shanghai Medpep Co.
Ltd. (China).
Synthetic chemistry. The conjugates were syn-
thesized by the Pharmaceutical Chemistry Institute
of Pharmacy College, Shandong University. Many
researchers have reported that the 2’ position of pa-
clitaxel is a suitable site for reversible derivatization
[18–20]. Structure-activity studies on pacliatxel re-
vealed that the C-13 ester side chain and its 2’-hydroxl
group appear essential for tubulin binding [13]. This
information suggests that the 2’ and 7’ position are the
most suitable sites for reversible derivatization [21].
2’-succinyl-paclitaxel (WP-06-1) was synthesized by
the procedure described by Cavallaro et al [20]. It was
activated by PyBOP and then coupled to octreotide.
Two kinds of conjugate were purified by high-perfor-
mance liquid chromatography (HPLC). WP-06-2A was
the broad singlet product purified by HPLC (HPLC.
15.67 min.; MS. 1998.7; MW. 1996.27) and WP-06-2B
was a pure singlet product purified by HPLC (HPLC.
20.85min.; MS. 2892.8; MW. 2890.19).
Cell culture. A549 cells were cultured in RPMI-1640
that contained 10% FBS. Calu-6 cells were cultured in
15% FBS. Fibroblast cells were cultured in RPMI-1640
medium that contained 20% fetal bovine serum. All
culture media were replaced with fresh media every
two to three days. The cells were cultured at 37 °C in
5% CO2 /95% air and were used when they were in
their growth phase.
the detection of SStr mrnA by rt-PCr.
The expression of SSTR mRNA using RT-PCR was
performed according to the manufacturer’s instruc-
tions. Total RNA was extracted using TRIzol reagent.
An aliquot (1 μg) of total RNA was reverse transcribed
using AMV-reverse transcriptase and then amplified
using specific primers for SSTR1, SSTR2, SSTR3,
SSTR4 and SSTR5 (Table 1). β-Actin was used as the
internal control. The PCR conditions were as follows:
(I) denaturing for 40 s at 94 °C; (II) annealing for 40 s
at 55 °C for SSTR2 and SSTR5 or for 40 s at 57 °C for
SSTR1, SSTR3 and SSTR4; (III) extension for 1 min
at 72 °C; and (IV) final extension for 5 min at 72 °C
cycled 30 times. The PCR products were visualized by
ethidium bromide staining and electrophoresis in 2%
agarose. The optical density (OD) of the target band
was corrected for the corresponding β-actin band. The
results were expressed as the OD ratio.
Table 1. RT-PCR Primers
Index Primers Length(bp)
SSTR-1 Forward 5’-ATGGTGGCCCTCAAGGCCGG-3’
Reverse 5’-CGCGGTGGCGTAATAGTCAA-3’ 318
SSTR-2 Forward 5’-TCCTCTGGAATCCGAGTGGG-3’
Reverse 5’-TTGTCCTGCTTACTGTCACT-3’ 332
SSTR-3 Forward 5’-GGAGAAGACTGAGGAGGAGG-3’
Reverse 5’-TTTCCCCAGGCCCTACAG-3’ 235
SSTR-4 Forward 5’-ATCTTCGCAGACACCAGACC-3’
Reverse 5’-ATCAAGGCTGGTCACGACGA-3’ 321
SSTR-5 Forward 5’-CGTCTTCATCATCTACACGG-3’
Reverse 5’-GGCCAGGTTGACGATGTTGA-3’ 223
β-actin Forward 5’-ATCATGAAGTGTGACGTGGAC-3’
Reverse 5’-AACCGACTGCTGTCACCTTCA-3’ 461
Cell proliferation assay. Cell proliferation was
assessed by the MTT-based cytotoxicity assay. Cells
were seeded at 4000 cells/well in 96-well plates, and
the protocol of the MTT assay was the same as that
described by Rivera et al. [22].
To investigate whether WP-06-1 retains the toxicity
of paclitaxel, we first treated A549 cells with different
concentrations (0.01, 1, 100, 1000 and 10 000 nmol/L)
of WP-06-1 and paclitaxel for 24 h. The cytotoxicity of
the conjugates in A549 cells and Calu-6 cells was then
assessed using concentrations of 0.01, 1, 100 and
1000 nmol/L conjugates for 24 h, 48 h and 72 h.
To determine whether WP-06-2A and WP-06-2B
cause cytotoxicity by binding to SSTRs, A549 cells
were pretreated with 10μmol/L octreotide for 30min
before 1μmol/L conjugates were added. It is known
that fibroblasts are low-SSTR-expressing cells [23, 24].
Therefore, we chose these cells as the SSTR-negative
control cells. The fibroblasts were treated by different
concentrations (0.01, 1, 100, and 1000 nmol/L) of
conjugates and paclitaxel for 24 h.
Cell-cycle analysis. Cell cycle perturbations were
determined after a 24 h treatment of 1 μmol/L pacli-
taxel, WP-02-A, WP-02-B by flow cytometry using a
Fluorescence-Activated Cell Sorter (FACS) (FC500,
Beckman Coulter Co. Ltd., USA).
Statistical analysis. SPSS statistical software
(version 12.0) was used for analyses. Statistical signifi-
cance was determined using the analysis of variance
(ANOVA). Data are expressed as mean ± standard
deviation (SD). Data were deemed statistically sig-
nificant at p < 0.05.
RESuLTS
Expression of SStrs in A549 cells, Calu-6 cells
and fibroblasts. The expression of mRNA for SSTR1,
SSTR2, SSTR4 and SSTR5 was detected in A549 and
Calu-6 cells. In the A549 cell line, the expression of
SSTR2 mRNA was conspicuous, whereas the expres-
sion of SSTR5 mRNA was weak. The expression of
SSTR5 mRNA in Calu-6 cells was obvious, but the
expression of SSTR2 mRNA was not marked (Fig. 1).
Neither cell line expressed SSTR3 mRNA nor mRNA
was detected in fibroblasts for any subtype of SSTR.
188 Experimental Oncology 29, 186–191, 2007 (September)
fig. 1. The detection of mRNA for SSTRs in A549 (a) and Calu-6
(b) cells by RT-PCR. Calu-6 and A549 cells did not express the
mRNA for SSTR-3. Lane M, marker; Lane 1, SSTR-1; Lane 2,Lane M, marker; Lane 1, SSTR-1; Lane 2,
SSTR-2; Lane 3, SSTR-3; Lane 4, SSTR-4; Lane 5, SSTR-5;
Lane 6, β-actin-actin
toxicity of the WP-06-1. There were no diffe-
rences in the cytotoxic activity of WP-06-01 and placli-
taxel in A549 cells (Table 2). This result established
that the succinyl group on the 2’ position of paclitaxel
is not essential for its toxicity. WP-06-1 retains the
toxicity of paclitaxel.
Table 2. Cell viability of A549 cells after a 24 h treatment of WP-06-1 and
paclitaxel (%, mean ± SD)
Concentration (nmol/L)
0 0.01 1 100 1000 10 000
Paclitaxel 100 84.4 ±
5.3
82.5 ±
3.1
73.6 ±
6.7
64.9 ±
6.3
69.2 ±
5.6
WP-06-1 100 85.3 ±
1.3
82.5 ±
6.2
70.3 ±
4.8
72.9 ±
5.1
65.2 ±
2.2
toxicity of the conjugates in SStr-positive
cells. The conjugates inhibited the growth of A549
and Calu-6 cells in a concentration-dependent man-
ner (Fig. 2, a and b). WP-06-2B was significantly more
cytotoxic than WP-06-2A and paclitaxel at low con-
centrations (0.01, 1 and 100 nmol/L) in A549 cells (p <
0.05). At 10 μmol/L, all the drugs caused precipitates
in the culture media. There were no differences in the
viability of A549 cells when treated with 1 μmol/L or
10 μmol/L paclitaxel. However, 10 μmol/L WP-06-2B
and WP-06-2A were more cytotoxic than that observed
at 10 μmol/L paclitaxel in A549 cells (p < 0.05). At this
concentration, the viability of A549 cells was 45.7 ±
6.9% with WP-06-2A, 46.7 ± 9.0% with WP-06-2B
treatment whereas for paclitaxel, viability was 69.2 ±
5.6% (see Fig. 2, a). In Calu-6 cells, the two conjugates
and paclitaxel caused concentration-dependent cyto-
toxicity to the same degree (see Fig. 2, b).
The conjugates also inhibited the growth of A549
and Calu-6 cells in a time-dependent manner (Fig. 2, c
and 2, d). In A549 cells, 100 nmol/L WP-06-2B showed
higher cytotoxicity than WP-06-2A and paclitaxel at
24 h. When the exposure times to the conjugates and
paclitaxel were extended to 48–72 h, there were no
differences in the relative viability or cytotoxicity in
A549 cells (see Fig. 2, c). In Calu-6 cells, 100 nmol/L
WP-06-2B showed less cytotoxicity than WP-06-2A
and paclitaxel at all exposure times (see Fig. 2, d).
fig. 2. The conjugates inhibited the growth of A549 and Calu-6
cells. Figures a and b show that 24 h exposure of A549 cells (a)
and Calu-6 cells (b) to the conjugates inhibited cell growth in a
concentration-dependent manner. Data of treatment with higher
concentrations are not shown, due to precipitation in cell media.
Figures c and d show the time-dependent inhibitory effect of
100 nmol/L conjugates on the growth of A549 (c) and Calu-6 (d)
cells. (Mean ± SD. *p < 0.05 vs PTX; ∆p < 0.05 vs WP-06-2A)
Octreotide modified the cytotoxicity of WP-06-2A and
WP-06-2B, but ameliorate the cytotoxicity of paclitaxel.
Following 24 h exposure to 1 μmol/L WP-06-2A, the vi-
ability of A549 cells was 55.4 ± 9.8%. Pretreatment for
30 min with 10 μmol/L octreotide increased the viability to
63.7 ± 5.4%. A similar result was seen when 1 μmol/L WP-
06-2B was used. Pretreatment with octreotide increased
cell viability from 55.8 ± 6.1% to 71.8 ± 9.6%. In contrast,
Experimental Oncology 29, 186–191, 2007 (September) 18929, 186–191, 2007 (September) 189September) 189) 189 189
pretreatment with octreotide exacerbated the cytotoxic
effects of 1 μmol/L paclitaxel (59.2 ± 6.4% vs 64.9 ±
6.3%; p < 0.05) (Table 3). Pretreatment of octreotide
therefore ameliorates the cytotoxicity of the conjugates
but potentiates the cytotoxicity of paclitaxel.
Table 3. Cell viability of A549 cells after a 24h treatment with/without
10 μmol/L octreotide pretreated for 30 min (%, mean ± SD)
Control Paclitaxel WP-06-2A WP-06-2B
Non-pretreated 100 64.9 ± 6.3 55.4 ± 9.8 55.8 ± 6.1
OCT pretreated 87.9 ± 9.9 59.2 ± 6.4* 63.7 ± 5.4* 71.8 ± 9.6*
*p < 0.05, vs Non-pretreated.
toxicity of the conjugates in fibroblasts
(SStr-negative) cells. Changes in cell morphology
were observed in fibroblasts following 24 h exposure to
1 μmol/L paclitaxel (data is not shown). These changes
were associated with a reduction in cell viability (64.7 ±
8.6%). Exposure to 1 μmol/L conjugates for 24 h
caused no overt changes in cell morphology, but was
associated with moderate reductions in cell viability
(WP-06-2A, 86.1 ± 1.8%; WP-06-2B, 90 ± 5.6%).
The toxicity of the conjugates on fibroblast cells was
significantly lower (p < 0.05) than that of paclitaxel.
Analysis of cell cycle. The G2/M ratio increased
from 13.4% in untreated A549 cells to 44.9% in A549
cells exposed to 1 μmol/L paclitaxel for 24 h (Fig. 3).
Exposure of these cells to 1 μmol/L of two conjugates
for 24 h increased this ratio further (WP-06-2A, 55.8%;
WP-06-2B, 75.4%) (see Fig. 3).
fig. 3. The cell cycle perturbations of A549 cells determined us-
ing FACs Scan flow cytometry after a 24 h exposure to 1 μmol/L
paclitaxel, WP-06-2A and WP-06-2B. Control (Con) represents
the results of A549 cells that were not exposed to any drugs
DIScuSSION
Over the past decade, several analogs of soma-
tostatin have been developed for the targeted delivery
of chemotherapeutic agents and antisense peptide
nucleic acids [25–28]. In doing so, these recent
advances in the synthesis and evaluation of such
compounds have contributed to the establishment
of the concept of targeted drug therapy. Remarkable
progress has also been made on the identification of
tumors and their metastases that express somatostatin
receptors. Current preclinical data provide good evi-
dence that SSTR-positive tumors can be targeted suc-
cessfully in vivo using somatostatin analogs coupled
to chemotherapeutic agents, which are more effective
and considerably less toxic than unconjugated cyto-
toxic radicals [29]. The anti-tumor drug, paclitaxel is
therapeutically beneficial for NSCLC. However, it is not
cell-specific, which is a major drawback. Therefore,
conjugates of octreotide and paclitaxel may be useful
for targeted drug therapy for NSCLC.
Several investigators have reported that human
NSCLC may overexpress SSTRs [5, 6]. However, the
expression of SSTRs by NSCLC cell lines remains con-
troversial. In agreement with the results of Ferone et al
[7], we detected mRNA for SSTR-2 and SSTR-5 in Calu-
6 cells and the expression of SSTR-5 was greater than
that of SSTR-2. We found mRNA for SSTR1 and SSTR4
in Calu-6 cells. Nayak et al [17] reported that A549 cells
do not express SSTRs. Given this result, we intended
to use A549 cells as a SSTR-negative control cell line.
However, we established that these cells expressed all
the subtypes of SSTR except SSTR-3. Furthermore,
we found that the expression of SSTR-2 was more pro-
nounced than that of SSTR-5. As a result, we had two
cells that we could use to evaluate the targeted delivery
of paclitaxel using an octreotide carrier.
In our experiment, WP-06-2A and WP-06-2B inhib-
ited the growth of A549 and Calu-6 cells in a concentra-
tion- and time-dependent manner and the suppression
of cell growth was potentiated at the higher concentra-
tions. When the exposure time was extended to 72 h,
the viabilities of A549 and Calu-6 cells were extremely
low. FACS analysis showed that the G2/M ratio in A549
cells in the presence of 1 μmol/L paclitaxel or the conju-
gates for 24 h increased. The result indicated that both
WP-06-2A and WP-06-2B maintained the cytotoxicity
of paclitaxel. Within our experiment, we showed that
SSTR-negative fibroblasts, WP-06-2A and WP-06-2B
were less cytotoxic than paclitaxel. When A549 cells
were pretreated with excess octreotide, the cytotoxici-
ties of both WP-06-2A and WP-06-2B decreased and
the cytotoxicity of paclitaxel increased. These results
show that WP-06-2A and WP-06-2B can bind selec-
tively to SSTRs and cause cell death specially [25, 29].
Because WP-06-2A and WP-06-2B were cytotoxic in
tumor cells expressing SSTRs, we suggest that the
development of paclitaxel-octreotide conjugates may
be valuable for targeted therapy of NSCLC.
The different expressions of SSTRs influenced the
cytotoxicity of WP-06-2B. In A549 cells, which overex-
190 Experimental Oncology 29, 186–191, 2007 (September)
press SSTR-2, the cytotoxic potency of WP-06-2B was
greater than that of paclitaxel at low concentrations. In
Calu-6 cells, which overexpress SSTR-5, 100 nmol/L
WP-06-2B was less cytotoxic than paclitaxel. How-
ever, the different expressions of mRNA for SSTR-2
and SSTR-5 have little influence on the cytotoxicity of
WP-06-2A. When compared to the cytotoxic effects
of free paclitaxel, the cytotoxic effects of WP-06-2A
were similar in A549 and Calu-6 cells, but were less
toxic in fibroblasts. Culler et al [30] reported that the
affinity of octreotide for SSTR-2 is higher than its
affinity to SSTR-5. Therefore, the affinity of WP-06-
2B for SSTR-2 might be greater than its affinity for
SSTR-5. This difference in affinity could account for
the different cytotoxicity of WP-06-2B in A549 and
Calu-6 cells because of the different expressions of
mRNA for SSTR-2 and SSTR-5 in the two cell lines.
When tumor tissue over-expresses mRNA for SSTR-2,
one might expect that WP-06-2B would have more
potent antitumor effects and less toxic side effects
than WP-06-2A. Further investigations are needed to
assess the biological properties of WP-06-2B in vivo.
There was an interesting result in our experiment.
All the drugs caused precipitates in cell culture media
when 10 μmol/L concentrations were used. The vi-
ability of A549 cells that were exposed to 10 μmol/L
paclitaxel was the same as that of A549 cells treated
with 1 μmol/L paclitaxel. Yet, the viabilities of A549
cells that were exposed to 10 μmol/L WP-06-2A and
WP-06-2B progressively decreased and were lower
than those of cells exposed to 10 μmol/L paclitaxel.
It is known that the aqueous solubility of paclitaxel is
extremely low [20] and octreotide is water-soluble [20,
31]. Therefore, the higher cytotoxicity of the conju-
gates might be due to their being more water- solubility
than paclitaxel in aqueous cell culture medium.
Schally et al. [10, 32] have developed a cytotoxic
analog of somatostatin, AN-238, in which the soma-
tostatin carrier peptide RC-121 was linked to 2-pyrro-
lino-doxorubicin (AN-201), which is a potent derivative
of doxorubicin. When compared to AN-201, they found
that AN-238 was more effective and less toxic in a
NSCLC H-838 xenogenic graft in nude mice. Moody
et al [8] synthesized camptothecin-somatostatin con-
jugate by linking camptothecin to the amino terminal of
somatostatin analog and reported that the conjugate
inhibited the growth of human small cell lung cancer
cells. Huang et al. [25] synthesized octreotide-con-
jugated paclitaxel by coupling paclitaxel succinate to
the amino terminal of octreotide. When comparing the
effects of free paclitaxel in human breast MCF-7 car-
cinoma cells, they showed that octreotide-conjugated
paclitaxel retained the biological activity of paclitaxel by
inducing the formation of tubulin bundles and eventually
causing apoptosis. All of our experimental results were
obtained using cultured cell systems. Given our results,
further research on in vivo cell selectivity and cytotoxic-
ity of WP-06-2A and WP-06-2B is warranted.
In conclusion, the results of this study show that
NSCLC cell lines, such as A549 and Calu-6 cells, ex-
press SSTRs. Irrespective of whether paclitaxel suc-
cinate was coupled to the amino acid end and/or the
free amino terminus of lysine of octreotide, WP-06-2A
and WP-06-2B were potent inhibitors of cell growth of
NSCLC cells in vitro. These results suggest strongly that
conjugates of somatostatin analogs and paclitaxel could
be used as efficacious agents for selective targeted
cancer chemotherapy in vitro and could be a promising
therapeutic modality for the treatment of NSCLC.
AcKNOwLEDgEMENTS
Project supported by the Science and Tech-
nology Commission of Shandong Province
(No. 2004GG3202017). The authors wish to thank
Dr. Bin XU for the supply of fibroblast cells and Dr. Jian-
min YANG for his assistance on the statistical analysis
of the data.
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Copyright © Experimental Oncology, 2007
КОНЪЮГАТЫ ПАКЛИТАКСЕЛА-ОКТРЕОТИДА ИНГИБИРУЮТ
РОСТ КЛЕТОК НЕМЕЛКОКЛЕТОЧНОГО РАКА ЛЕГКОГО in vitro
Цель: оценить эффект конъюгатов паклитаксела-октреотида на рост культивированных клеток немелкоклеточного рака
легкого человека. Методы: для определения мРНК подтипов рецептора соматостатина человека (SSTR) применяли ОT-ПЦР.
Анализ цитотоксичности в МТТ-тесте применяли для оценки выживаемости клеток после их инкубации с паклитакселом
и конъюгатами. Нарушения клеточного цикла определяли с применением FACS — клеточного сортера. Результаты:
установлено, что клеточные линии немелкоклеточного рака легкого A549 и Calu-6 экспрессируют SSTR2 и SSTR5 мРНК.
Отмечено эффективное дозо- и времязависимое угнетение роста клеток A549 и Calu-6 паклитакселом и конъюгатами.
Для SSTR-негативных фибробластов конъюгаты менее цитотоксичны, чем паклитаксел. Конъюгаты и паклитаксел могут
индуцировать повышение соотношения фаз G2/M в клетках A549. Выводы: конъюгаты паклитаксел-октреотида могут быть
использованы как селективные химиотерапевтические агенты для воздействия на немелкоклеточный рак легкого.
Ключевые слова: немелкоклеточный рак легкого, октреотид, паклитаксел.
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